Impact of radiation processing on some phytochemical ...

Version approved during the meeting of Deans held during 29-30 Nov 2013

Impact of radiation processing on some

phytochemical constituents of selected Indian vegetables and their products

By

JYOTI TRIPATHI

CHEM01200904006

Bhabha Atomic Research Centre, Mumbai

A thesis submitted to the

Board of Studies in Chemical Sciences

In partial fulfillment of requirements

for the Degree of

DOCTOR OF PHILOSOPHY

of

HOMI BHABHA NATIONAL INSTITUTE

February, 2015


STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an

advanced degree at Homi Bhabha National Institute (HBNI) and is deposited in the

Library to be made available to borrowers under rules of the HBNI.

Brief quotations from this dissertation are allowable without special permission,

provided that accurate acknowledgement of source is made. Requests for permission

for extended quotation from or reproduction of this manuscript in whole or in part

may be granted by the Competent Authority of HBNI when in his or her judgment the

proposed use of the material is in the interests of scholarship. In all other instances,

however, permission must be obtained from the author.

Jyoti Tripathi


DECLARATION

I, hereby declare that the investigation presented in the thesis has been carried out by

me. The work is original and has not been submitted earlier as a whole or in part for a

degree / diploma at this or any other Institution / University.

Jyoti Tripathi


Dedicated to “The Almighty”


ACKNOWLEDGEMENTS

My heartiest gratitude for the Almighty, who made me to see this day! This

dissertation is the result of efforts from several people. First and foremost, I would

like to express my sincere gratitude to my thesis advisor Dr. P. S. Variyar for his

continuous support to my work, patience, encouragement, and immense knowledge.

Without his supervision and constant support this project would not have been

possible. It is also my great pleasure to thanks Dr. A. K. Sharma (former Head, FTD)

and Dr. S. P. Kale, Head, FTD, for their encouragement and support.

I would like to express my gratitude to the members of my doctoral committee, Dr. S.

Chattopadhyay, Dr. K. I. Priyadarsini and Dr. S. Banerjee for their generous support

and constructive suggestions. I also owe a great debt of gratitude to my collaborators

Dr. S. Jamdar, Dr. S. K. Ghosh, Dr. A. K. Bauri, and Dr. T. R. Ganapathi for their

help and fruitful discussions during the course of the work.

My heartiest gratitude and thanks are due to my colleagues and friends in FFACS Dr.

S. Chatterjee, Sumit Gupta, Jasraj, Vanshika, Prashant, Rupali, Vivekanand, Snehal,

Aparajita, Chaturbhuj and Sarver for their companionship, invaluable help and

motivation throughout the work, which kept me going.

I would like to thank my parents and parents-in-law, for their unconditional love,

support, faith, encouragement and confidence in me. They have been my driving force

and inspiration in all pursuits of life. I would also like to thank my sisters-in-law and

brothers for constant encouragement and support. I run short of words in expressing

my gratitude towards my beloved life partner Rahul, who acted as an anchor in all the

waves of hardships. Finally, I would take this opportunity to thank all my teachers,

who made me what I am today.

Jyoti

i

CONTENTS

CONTENTS Page No.

SYNOPSIS ix-xx

LIST OF FIGURES xxi-xxv

LIST OF TABLES xxvi-xxix

Table of Contents

Chapter 1. Introduction…………………………………………………...........1-67

1. Importance of vegetables in the diet 2

1.2. Minimal processing of vegetables 5

1.2.1. Microbial spoilage 7

1.2.2. Appearance and color 8

1.2.3. Texture 10

1.2.4 Flavor (taste and aroma) 12

1.2.4.1. Taste 12

1.2.4.2. Aroma constituents 13

1.2.5. Nutritional value 17

1.2.5.1. Carotenoids 17

1.2.5.2. Phenolics 19

1.2.5.2.1. Flavonoids 19

1.2.5.2.2. Phenolic acids 22

ii

1.2.5.3. Glucosinolates 23

1.2.5.4. Triterpenoids 25

1.3. Quality measurement of fresh-cut produce - Sensory and

instrumental measurements

27

1.3.1. Sensory methods of quality measurement 27

1.3.2. Instrumental methods of quality measurement 29

1.3.2.1. Color 30

1.3.2.2. Texture 32

1.3.2.3. Flavor- Aroma and Taste 33

1.3.3 Analysis of bioactive phytochemicals 40

1.4 Different methods for preservation of fresh-cut produce 41

1.4.1 Chemical preservatives 42

1.4.2. Physical methods of preservation 43

1.4.2.1. Radiation Processing 44

1.4.2.2. Advantages of Radiation Processing 47

1.4.2.3. Effect of gamma irradiation on shelf-life of RTC vegetables 48

1.4.2.4. Microbial status of irradiated RTC vegetables 49

1.4.2.5. Effect of radiation processing on phytochemicals 50

1.5. Vegetables selected for the present work 52

1.5.1. Ash gourd (Benincasa hispida) 52

1.5.2. Drumstick (Moringa oleifera) 56

1.5.3. Pumpkin (Cucurbita pepo) 61

iii

1.6. Scope of the work - Aims and objectives 65

Chapter 2. Materials and methods…………………………………………69-108

2.1. Materials 70

2.1.1. Plant material 70

2.1.2. Chemicals and materials 70

2.2. Methodology 71

2.2.1. Development of ready-to-cook (RTC) vegetables using

radiation processing

71

2.2.1.1. Preparation of RTC vegetables 71

2.2.1.2. Radiation processing and storage 72

2.2.1.3. Optimization of quality parameters 72

2.2.2. Characterization of the developed products at optimized

parameters

76

2.2.2.1. Nutritional quality 76

2.2.2.2. Aroma quality 78

2.2.2.2.1. Isolation and identification of free aroma compounds 78

2.2.2.2.2. Isolation and identification of bound aroma precursors 82

2.2.2.2.3. Chemometric analysis of the aroma data 86

2.2.2.2.4. Identification of aroma impact compounds 86

2.2.2.3. Headspace gas composition inside packages 89

2.2.2.4. Gamma irradiation induced browning inhibition 89

2.2.2.4.1. Isolation and identification of phenolic compounds 89

iv

2.2.2.4.2. Effect of radiation processing on enzymes involved in

browning

90

2.2.2.4.3. Scanning Electron Microscopic (SEM) analysis 93

2.2.2.4.4. Electrolytic leaching 93

2.2.2.4.5. Statistical analysis 93

2.2.3. Isolation, identification and studying the effect of radiation

processing on bioactive phytochemicals

94

(A) Ash gourd

2.2.3.1. Compounds with plant growth promoting activities 94

2.2.3.1.1. Preparation of ash gourd extracts 94

2.2.3.1.2. Activity Guided Fractionation 95

2.2.3.1.3. Structure Identification 97

2.2.3.1.3.1. Spectral Studies 97

2.2.3.1.3.2. Chemical synthesis of acetoin glucoside 98

2.2.3.1.4. Growth promotion assay of purified acetoin glucoside 99

2.2.3.1.5. Protein Extraction and Two-Dimensional Gel Electrophoresis 99

2.2.3.1.6. Mass Spectrometric analysis and Database Searching 100

2.2.3.1.7. Statistical Analysis 100

2.2.3.2. Compounds with ACE inhibition activity 100

2.2.3.2.1. Preparation of ash gourd extracts 100

2.2.3.2.2. Preparation of enzyme extract 101

2.2.3.2.3. ACE inhibition assay 101

v

2.2.3.2.4. Activity guided fractionation and identification of active

principles

102

(B) Pumpkin 103

2.2.3.3. Isolation and identification of phenolic constituents 103

2.2.3.4. Isolation and identification of carotenoids 103

2.2.3.5. Isolation and identification of triterpenes 104

(C) Drumstick

2.2.3.6. Isolation, identification and quantification of phenolic

constituents

106

2.2.3.7. Isolation and identification of glucosinolates 106

Chapter 3. Results and discussion……………………………………….109-274

3.1. Development of radiation processed shelf stable RTC

vegetables

110

3.1.1. Optimization of radiation dose and storage period with

mathematical modeling

110

3.1.1.1. Microbial quality 120

3.1.1.2. Color 125

3.1.1.3. Texture 128

3.1.1.4. Sensory acceptability 131

3.1.1.5. Optimization and verification of results 133

3.2. Characterization of the developed RTC products 137

3.2.1. Sensory quality evaluation by Quantitative Descriptive 138

vi

Analysis (QDA)

3.2.2. Nutritional quality 141

3.2.2.1. DPPH Radical scavenging activity 142

3.2.2.2. Total phenolic and flavonoid content 144

3.2.2.3. Vitamin C content 147

3.2.3. Headspace gaseous composition 150

3.2.4. Aroma quality - chemical analysis of free and bound aroma

compounds and identification of key odorants

153

3.2.4.1. Ash gourd 155

3.2.4.1.1. Free aroma analysis 155

3.2.4.1.2. Bound aroma analysis 160

3.2.4.1.3. Identification of key odorants 166

3.2.4.2. Drumstick 171




3.2.4.3. Pumpkin 188




3.2.5. Effect of radiation processing on free and bound aroma

profiles of the selected vegetables

204

vii

3.2.6. Gamma irradiation induced browning inhibition 218

3.2.6.1. Evaluation of parameters responsible for browning 219

3.2.6.1.1. Electrolytic leaching 219

3.2.6.1.2. Changes in micro-structure 221

3.2.6.1.3. Evaluation of enzyme activities 222

3.2.6.1.4. Analysis of phenolic constituents 225

3.2.6.1.5. Correlations between various factors contributing in browning

inhibition

227

3.2.6.1.6. Role of α-resorcylic acid in inhibition of ash gourd PPO -

Kinetics studies

228

3.3. Isolation, identification of bioactive constituents and

determining their changes during radiation treatment

232

3.3.1. Ash gourd 232

3.3.1.1. Plant growth promoting activity 233

3.3.1.1.1. Isolation and identification of Plant Growth Regulating

Compound

233

3.3.1.1.2. Growth Promotion Studies 235

3.3.1.1.3. Identification of differentially expressed protein in acetoin

glucoside treated tobacco leaf discs

239

3.3.1.2. Angiotensin converting enzyme (ACE) inhibition activity 242

3.3.1.2.1. Activity guided fractionation 243

3.3.1.2.2. Isolation and identification of active principles 244

viii

3.3.2. Cucurbitacins in pumpkin 248

3.3.2.1. TLC analysis 249

3.3.2.2. Identification of cucurbitacins 252

3.3.3. Glucosinolates in drumstick 256

3.3.3.1. Isolation and identification of glucosinolates and

isothiocyanates

257

3.3.4. Isolation, identification and quantification of phenolic

constituents and carotenoids

263

3.3.4.1. Ash gourd 265

3.3.4.2. Drumstick 266

3.3.4.3. Pumpkin 267

3.3.4.3.1. Identification and quantification of phenolic constituents 267

3.3.4.3.2. Identification and quantification of carotenoids 269

3.4. Conclusions 271

Chapter 4. Summary…………………………………………………………..275-279

References……………………………………………………………………...280-315

List of Publications 316-318

ix

SYNOPSIS

Vegetables are vital components of a healthy diet and provide essential nutrients and

bioactive phytochemicals. There are evidences that diets rich in vegetables can decrease

the levels of chronic diseases including cancer [1, 2]. Vegetables possess high

antioxidant properties that are imparted by bioactive compounds. These compounds

present in them protect bio-molecules from oxidative damage, thus playing a major

health protective role.

In recent years there has been an increase in demand for convenience foods such as

ready-to-cook (RTC) vegetables by consumers. Low shelf life of majority of these

products, however, restricts their marketability. Apart from microbial safety,

maintenance of fresh-like characteristics is the main criteria determining consumer

acceptability of the product. Various post-harvest processing techniques are currently

available that can kill pathogens while retaining fresh attributes of the produce.

Radiation processing is a promising technology for improving the shelf life of fresh-cut

produce. Treatment of food products by ionizing radiation is a physical process

involving direct exposure to electromagnetic γ-rays, X-rays or electron beam for

improvement in food safety and shelf life. It is a non-thermal technology that effectively

eliminates food-borne pathogens in various foods, including fresh vegetables without

compromising the nutritional properties or sensory qualities of food [3] Gamma

radiation being ionizing in nature causes radiolysis of water thereby producing reactive

hydroxyl radical. These radicals are extremely reactive and attack and damage cellular

components, especially DNA. Due to damage to genetic material there is inhibition in

x

microbial growth, thus inactivating microorganisms [4]. Unlike typically processed

foods, fresh-cut products consist of living tissues and post harvest processing treatments

including irradiation can act as stress bringing about change in post harvest physiology

of the product. The critical factors affecting consumer acceptability include microbial

and sensory quality (color, texture, flavor /aroma). Apart from the sensory attributes, the

bioactive constituents such as phenolic constituents, carotenoids etc. are mainly

responsible for the beneficial effects of vegetables. Existing literature suggests an

increased extractability and bioavailability of bioactive constituents as a result of

radiation processing [5]. An enhanced breakdown of bound precursors including aroma

and phenolic glycosides has also been reported. It can therefore be inferred that radiation

processing can enhance aroma by hydrolyzing aroma glycosides, increase total phenolics

and thus antioxidant capacity, while bringing about microbial decontamination of food

products. Ash gourd (Benincasa hispida), drumstick (Moringa oleifera) and pumpkin

(Cucurbita pepo) are traditional Indian vegetables used in Indian cuisine. These

vegetables have recently been marketed as RTC products due to the convenience they

offer. No reports exist so far on the impact of radiation processing on the above

vegetables or their RTC products. The present thesis aims at developing radiation

processed RTC products of the above vegetables with improved shelf-life and

understanding changes in bioactive compounds and their precursors during such a

treatment.

Chapter 1 of the thesis introduces the subject of food irradiation with special emphasis

on irradiation of minimally processed fresh-cut vegetables and describes the scientific

xi

literature related to the present work. Based on the review of available literature, it was

found that radiation processing leads to microbial decontamination thereby improving

the shelf-life of fresh-cut fruits and vegetables. It can also lead to increased contents of

various bioactive principles, mainly responsible for the beneficial effects of vegetables.

However, very few reports have dealt with the impact of radiation processing on the

postharvest quality of vegetables of Indian origin. The chapter provides general

information on vegetables with special reference to ash gourd, drumstick and pumpkin,

the three most widely used Indian vegetables. The current information on the chemical

aspects of these vegetables and general methods of isolation and identification of

chemical constituents, in particular, the bioactive constituents are detailed. Commonly

used methods for isolation and identification of aroma constituents of vegetables and

their quantification using instrumental methods such as GC/MS, HPLC are also

discussed.

Chapter 2 of the thesis describes the materials and experimental methods. The vegetable

samples of ash gourd, drumstick and pumpkin were obtained from local growers in and

around Mumbai, India. Irradiation was carried out using a food package irradiator (GC

5000, Board of Radiation and Isotope Technology, India) at BARC, Mumbai.

Gamma irradiation was used for shelf-life extension of RTC vegetables. Sensory quality

was assessed by a sensory panel through hedonic testing. Color was evaluated by

colorimeter and texture through texture analyzer according to the standard protocols.

Nutritional parameters like vitamin content, total phenolic content and antioxidant

properties were studied according to standard AOAC protocols. Activities of different

xii

enzymes were assayed as per reported spectrophotometric methods. The non-volatile

constituents that included phenolic acids, carotenoids and triterpenes were studied by

TLC and HPLC analysis.

Likens-Nikersons simultaneous distillation extraction (SDE) apparatus and solid phase

microextraction (SPME) techniques were used for isolation of free aroma volatiles.

Aroma glycosides were extracted using XAD and solid phase extraction (SPE) using C-

18 reverse phase cartridges. The total ion current obtained from SPME (for free aroma)

and SPE (for bound aroma) was further processed using chemometrics (PCA) for

analyzing the effect of radiation treatment on aroma constituents.

Chapter 3 deals with the results and discussion. It has been divided into following

subsections.

3.1. Development of radiation processed shelf-stable RTC products: Preliminary

screening of vegetables was done based on the feasibility of gamma irradiation for shelf-

life improvement. Effect of radiation processing (0.5-2.0 kGy) on the shelf life of five

RTC vegetables, namely ash gourd (Benincasa hispida), pumpkin (Cucurbita pepo),

bottle gourd (Lagenaria siceraria), lady finger (Abelmoschus esculentus) and drumstick

(Moringa oleifera) were studied when stored at 10 °C. In the case of ready-to-cook

(RTC) ash gourd, pumpkin and drumstick, radiation processed samples were acceptable

up to storage duration of 10-25 d depending on the vegetable and radiation dose

delivered. Cut bottle gourd pieces developed browning/ blackening after a few minutes

of cutting making them unacceptable. In case of ladies finger, the treated and untreated

samples were acceptable only up to a storage period of 1-2 d, beyond which increased

xiii

sliminess and off odor in the radiation treated samples restricted their acceptance. Ash

gourd, drumstick and pumpkin were therefore selected for the development of radiation

processed shelf-stable products. The data obtained for microbial analysis, color, texture

and sensory acceptability (hedonic analysis) were separately fitted in cubic polynomial

equations for each vegetable. Optimum radiation dose and storage period for each

product was determined by solving the model equations using criteria (total mesophilic

counts < 5 log CFU/g; overall sensory acceptability > 5 with minimum change in color

and texture). For ash gourd, optimum solution was a shelf life of 12 d at a radiation dose

of 2 kGy, while for drumstick and pumpkin, the optimum parameters were a shelf life of

12 and 21 d respectively at a radiation dose of 1 kGy.

3.2. Characterization of the developed products: The radiation processed products

were characterized for various physical and chemical parameters important for consumer

acceptability. Sensory analysis by quantitative descriptive analysis (QDA) indicated that

radiation processed products possessed excellent sensory quality at the end of intended

storage period. An appreciable increase in DPPH radical scavenging activity was

observed as a result of radiation treatment in all the three vegetables which was linearly

correlated with increase in total phenolic and flavonoid contents.

Vegetables impart characteristic flavor (aroma and taste) to the cuisine. In the green

form, aroma of majority of the vegetables is indistinguishable from each other. Cooking

results in liberation of their characteristic aroma from their precursors. Very few reports

however exist on the nature of these precursors in vegetables, particularly of Indian

origin. The free and glycosidically bound aroma compounds of ash gourd, drumstick and

xiv

pumpkin were studied. Despite of the presence of several volatile aroma compounds in a

food matrix, not all of them are responsible for the characteristic odor of a food product.

Therefore there is a great interest in determining the contribution of each constituent

towards the overall flavor of a product. Key odorants of each vegetable were therefore

characterized. GC-O analysis of the aroma extracts was successfully employed for the

identification of key odorants in the vegetables studied. Acetoin, octanal and nonanal in

ash gourd; benzothiazole, decanal and 2E-decenal in drumstick and a combination of 6Z-

nonenal and 2E, 6Z-nonadienal in case of pumpkin were identified as the key odorants

responsible for the characteristic aroma. Further, the effect of radiation processing and

storage on aroma composition was examined. The GC/MS data obtained for free and

bound aroma was subjected to Principal Component Analysis (PCA) for each vegetable

separately. PCA is an unsupervised technique which is used for dimensionality reduction

of multivariate data sets and allows visualization of complicated data for easy

interpretation [6]. To know the nature of the constituents responsible for the differences

among control and radiation processed samples of different days, factor loading data was

analyzed. Contents of alcohols increased in response to radiation processing and storage

in ash gourd and pumpkin which could be attributed to the radiolytic breakdown of their

corresponding glycosidic precursors. Contents of major carbonyl compounds decreased

with radiation processing and storage in ash gourd, while a significant increase in the

content of hexanal and trans-2-hexenal was observed in radiation processed drumstick.

The increase in aldehyde contents could be attributed to radiation induced lipid

radiolysis resulting in release of linolenic acid and its subsequent conversion to

xv

aldehydes via lipoxygenase (LOX) pathway. On the other hand, conversion of aldehydes

to corresponding branched chain alcohols and subsequently to esters could be the reason

for decreased contents of aldehydes in ash gourd.

A considerable difference in the effect of radiation processing on the content of

glycosidic precursors was observed among the three vegetables studied. Two types of

radiation effects were observed. Decrease in the content of these precursors was noted in

ash gourd and pumpkin, while the contents increased in irradiated drumstick. Both

radiation induced increased extractability and degradation was observed. It can be

concluded that both these changes can occur simultaneously and independent of each

other. Despite the changes in the content of some of the aroma compounds during

storage and radiation processing as noted instrumentally and statistically, they were not

sufficient to be observed by the sensory panel. Hence the radiation processed product

developed, had good sensory acceptability at the end of storage period.

Gamma-radiation (2 kGy) induced inhibition of browning in RTC ash gourd stored

(10°C) up to 12 d was further investigated. In the control samples, phenylalanine

ammonia lyase (PAL) activity increased during storage that could be linearly correlated

with enhanced quinone formation and browning. Radiation treatment resulted in a

significant increase in the content of alpha resorcylic acid, a known PPO inhibitor. The

decreased PPO activity was thus correlated with the increased content of this acid in

irradiated samples. The kinetic parameters of α-resorcylic acid inhibition were

determined using Linweaver-Burk plots. The nature of inhibition was found to be mixed

and reversible type. No significant change was observed in peroxidase activity. So

xvi

browning inhibition in radiation processed (2 kGy) RTC ash gourd during storage could

be a synergistic effect of decreased quinone formation and enzyme (PPO and PAL)

activities.

3.3. Isolation, identification of bioactive phytochemicals and determining their

changes during radiation processing

3.3.1. Ash gourd

3.3.1.1. Plant growth promoting activity: The ash gourd waste (peel and seeds) has

been traditionally used as a green manure for increasing the soil nutrients while shelled

seeds are reported to have anabolic properties that promote tissue growth. The vegetable

extract was therefore screened for the bioactive principles responsible for the plant

growth promoting activity. Ash gourd juice was fractionated with solvents of different

polarities (ether, ethyl acetate and n-butanol) and the butanol fraction was analyzed by

TLC and HPLC. The major compound in this fraction was purified by preparative TLC

and identified as acetoin glucoside based on NMR and mass spectral studies as well as

by chemical synthesis. In vitro treatment by soaking tobacco leaf discs in MS liquid

medium containing different concentrations of this compound in the range of 0.044-0.88

mg resulted in a significant increase in the diameter of leaf discs. At an optimum

concentration of 0.176 mg, the diameter of leaf discs increased to 1.13 cm compared to

0.6 cm in control. Two dimensional gel electrophoresis of the proteins isolated from the

untreated and acetoin glucoside treated tobacco leaf discs revealed the expression of a

new protein in the treated samples. This was identified as a Ras related nuclear GTPase

or Ran protein, a very important regulatory protein, by peptide mass finger printing. The

xvii

study demonstrated for the first time the role of acetoin glucoside as the compound

responsible for the tissue growth regulating properties of the vegetable. The content of

acetoin glucoside, the active principle, remained unaffected in radiation-treated ash

gourd. Thus the plant growth activity was maintained in the irradiated samples.

3.3.1.2. Angiotensin converting enzyme (ACE) inhibition activity: The juice of ash

gourd is recommended to patients suffering from heart ailments and high blood pressure.

The ash gourd juice was fractionated with solvents of different polarity (ether, ethyl

acetate and water) and each of them was screened for ACE inhibition activity by an

enzymatic assay using hippuryl-histidine-leucyl as substrate. The aq extract obtained

from non-irradiated control and irradiated ash gourd at a concentration of 50 mg/mL

exhibited 47 % and 41 % ACE inhibition respectively. This extract was purified by RP-

HPLC and further by gel filtration. The molecular weight of the active fraction as

determined by GPC was 323 Da. The purified fraction showed a UV absorbance at 220

and 254 nm. Mass spectral analysis showed the presence of amino acids viz. alanine

(M+, 89) and valine (M+, 117) in this fraction. This confirms the presence of a small

peptide comprising of these amino acids as the active principle. However, the compound

requires further characterization which is under progress. The ACE inhibition activity of

the ash gourd however remained unaffected due to irradiation.

3.3.2. Triterpenes in pumpkin: Cucurbita glycosides from pumpkin were isolated by

extracting with methanol and further purified by preparative TLC. The isolated

compounds were subjected to HPLC, where the peak at retention time 4.27 was

tentatively identified as cucurbitacin E glucoside. The total extract was enzymatically

xviii

hydrolyzed as well as acetylated and then subjected to GC/MS analysis. The mass

fragmentation of the identified peaks was compared with that of cucurbitacins reported

in literature. Based on this data, two major compounds were identified as Cucurbitacin C

and E. Thus based on the HPLC analysis of total extract and GC/MS analysis of

enzymatically hydrolyzed and acetylated extract the major triterpenes identified were

glycosides of Cucurbitacin C and E. Estimation of the active principles in control and

irradiated pumpkin by densitometer indicated a significant (p < 0.05) decrease in their

contents during storage. Increase in activity of hydrolytic enzymes such as glycosidase

and pectinase during storage in fruits and vegetables has been reported [5]. This could

account for the decrease in contents of these compounds during storage. However, the

contents of identified active principles were unaffected due to radiation processing.

3.3.3. Glucosinolates in drumstick: Effect of radiation treatment (1 kGy) on bioactive

glucosinolates in drumstick was investigated. Food processing methods result in their

hydrolysis and various breakdown products are formed which are generally identified

using GC/MS. Among breakdown products, isothiocyanates (ITCs) have the highest

biological activity. They have been reported to possess broad-spectrum antimicrobial

activity against bacterial, fungal pathogens and insects [7, 8] and possess potent

anticarcinogenic activity. Major isothiocyanates identified in drumstick were isopropyl

isothiocyanate (4.38 ng/g), 2-butyl isothiocyanate (1.99 ng/g) and isobutyl

isothiocyanate (2.35 ng/g). The content of all the three isothiocyanates increased in

response to radiation processing (1 kGy) with a 2-3 times higher content in radiation-

treated samples at the end of storage as compared to the respective non-irradiated

xix

samples. The observation suggests an improved nutraceutical value of the vegetable by

radiation processing.

3.3.4. Isolation, identification and quantification of phenolic constituents and

carotenoids: The phenolic constituents of all the three vegetables were extracted with

methanol and subjected to HPLC-DAD analysis. Syringic, chlorogenic and α-resorcylic

acid were identified as major phenolic constituents in ash gourd; protocatechuic, p-

hydroxybenzoic, vanillic acid and quercetin in drumstick while pumpkin was

characterized with the presence of gallic, caffeic, o-coumaric acid and kaempferol.

Detailed analysis revealed that radiation processing resulted in increased extraction of

phenolic constituents in ash gourd, while no significant effect of radiation processing

was observed on the phenolic constituents of drumstick and pumpkin.

Pumpkin is a good source of carotenoid pigments. Apart from imparting color, they act

as antioxidants and enhancers of the immune response. The carotenoids of pumpkin

were extracted in hexane and analyzed by HPLC-DAD. The major carotenoids identified

were lutein and β-carotene. The contents of both the constituents remained unaffected in

response to the radiation treatment. To the best of our knowledge, this study

demonstrates for the first time the effect of radiation processing on shelf life

improvement and on the phytochemical constituents of RTC ash gourd, drumstick and

pumpkin.

Finally the achievements of these studies will be highlighted in the conclusion section

that will follow chapter 3.

xx

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Mechanisms’, Cancer Causes Control, 427–42.

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sensory, and shelf life. In: Food irradiation research and technology. Editors: Sommers,

H. S. and Fan, X. Blackwell Publishing, U.S.A. pp.169– 184.

4. Arvanitoyannis, I. S., Stratakos, A. C., & Tsarouhas, P. (2009). Irradiation

applications in vegetables and fruits: a review. Critical reviews in food science and

nutrition, 49(5), 427-462.

5. Alothman, M., Bhat, R., & Karim, A. A. (2009). Effects of radiation processing on

phytochemicals and antioxidants in plant produce. Trends in food science & technology,

20(5), 201-212.

6. Eriksson, L., Johansson, E., Kettaneh-Wold, N., & Wold, S. (2001). Multi-and

megavariate data analysis: principles and applications. Umetrics.

7. Lin, C. M., Kim, J., Du, W. X., & Wei, C. I. (2000). Bactericidal activity of

isothiocyanate against pathogens on fresh produce. Journal of Food Protection, 63(1),

25-30.

8. Wittstock, U., Kliebenstein, D. J., Lambrix, V., Reichelt, M., & Gershenzon, J.

(2003). Chapter five glucosinolate hydrolysis and its impact on generalist and specialist

insect herbivores. Recent advances in phytochemistry, 37, 101-125.

xxi

LIST OF FIGURES

Figure Title Page No.

Figure 1. Reactions depicting mechanism of PPO action in browning development 10

Figure 2. Representative aroma compounds of vegetables 15

Figure 3. Structure of glycosidic portion of aroma glycosides 16

Figure 4. Some representative carotenoids found in vegetables 18

Figure 5. General structure of glucosinolates and their enzymatic degradation

products

25

Figure 6. Chemical structure of cucurbitane 26

Figure 7. Diagram depicting three dimensional L, a and b color space 31

Figure 8. Glucosinolates reported in drumstick (Moringa Oleifera 60

Figure 9. A schematic representation of the different methodologies adopted for

the isolation and analysis of free aroma volatiles

79

Figure 10. A schematic representation of the different procedures followed for

isolation and analysis of bound aroma precursors

83

Figure 11. Scheme of chemical synthesis of acetoin-3-O-β-D- glucoside 98

Figure 12. Variation of (a) Total plate counts and (b) yeast and mold counts with

radiation dose (0 – 2.5 kGy) and storage time (0 – 15 d) in RTC ash

gourd

121

Figure 13. Variation of (a) Total plate counts (b) yeast and mold counts with

radiation dose (0 – 2.5 kGy) and storage time (0 – 15 d) in RTC

drumstick

121

Figure 14. Variation of (a) Total plate counts and (b) yeast and mold counts with

radiation dose ( 0 – 2.5 kGy) and storage time (0 – 28 d) in RTC

pumpkin

122

Figure 15. Effect of radiation treatment and storage on (a) L values of RTC ash

gourd; (b) a values of RTC drumstick; (c) L values of RTC pumpkin

126

xxii

Figure 16. Effect of radiation treatment and storage on firmness of (a) Ash gourd;

(b) Drumstick; (c) Pumpkin

129

Figure 17. Effect of radiation treatment and storage on the overall sensory

acceptability of (a) Ash gourd; (b) Drumstick; (c) Pumpkin

131

Figure 18. Control and radiation processed (at optimum dose) RTC products for ash

gourd, drumstick and pumpkin at the end of intended storage period

137

Figure 19. Quantitative descriptive analysis of fresh control and radiation-processed

ash gourd samples at day zero and twelve of storage

139


drumstick samples at day zero and twelve of storage

140


pumpkin samples at day zero and 21 of storage

141

Figure 22. Variation in DPPH radical scavenging capacity of control and radiation

processed RTC vegetables with storage. (A) Ash gourd, (B) Drumstick,

(C) Pumpkin

143

Figure 23. Variation in total phenolic and flavonoid content in ash gourd (A & D);

drumstick (B & E) and pumpkin (C & F) due to radiation processing and

storage

145

Figure 24. Effect of radiation processing and storage on ascorbic acid content of (A)

Ash gourd; (B) Drumstick; and (C) Pumpkin

148

Figure 25. Headspace composition in control and radiation processed RTC (A) Ash

gourd (B) Drumstick and (C) Pumpkin packages during storage

151

Figure 26. GC/MS profiles of volatile aroma of ash gourd obtained using SDE,

HVD and SPME methods

156

Figure 27. GC/MS profile of free aglycones released from bound aroma precursors

of ash gourd obtained from XAD and SPE

161

Figure 28. Odor Detection frequency chromatogram of Ash gourd SDE volatiles 169

Figure 29. Chemical structures and mass spectra for most potent odorants of ash

gourd

170

xxiii

Figure 30. GC/MS chromatogram of cryotrapped fraction(4.5-5.5 min) isolated

corresponding to ash gourd aroma

170

Figure 31. GC/MS profiles of volatile aroma compounds of drumstick obtained

using SDE and SPME methods

171

Figure 32. XAD and SPE chromatograms of drumstick 177

Figure 33. Odor detection frequency chromatogram of drumstick SDE volatiles 184

Figure 34. TLC aromagram of drumstick SDE isolate 186

Figure 35. Chemical structures and mass spectra of most potent odorants of

drumstick

188

Figure 36. GC/MS profiles of SDE and SPME isolates of pumpkin 189

Figure 37. GC/MS profile of hydrolyzed aroma glycosides of pumpkin obtained

from XAD and SPE isolates

196

Figure 38. Odor detection frequency chromatogram of pumpkin aroma isolate 202

Figure 39. Chemical structures and mass spectra for most potent odorants of

pumpkin

203

Figure 40. PCA of free aroma of ash gourd (C0, C5, C8, C12 – control samples of

day 0, 5, 8 and 12; I0, I5, I8, I12 – irradiated (2 kGy) samples of day 0, 5,

8 and 12)

206

Figure 41. PCA of bound aroma of ash gourd (C0, C5, C8, C12 – control samples of


8 and 12)

207

Figure 42. PCA of free aroma of drumstick (C0, C5, C8, C12 – control samples of


8 and 12)

211

Figure 43. PCA of bound aroma of drumstick (C0, C5, C8, C12 – control samples

of day 0, 5, 8 and 12; I0, I5, I8, I12 – irradiated (1 kGy) samples of day

0, 5, 8 and 12)

212

xxiv

Figure 44. PCA of free aroma of pumpkin (C0, C7, C14, C21 – control samples of

day 0, 7, 14 and 21; I0, I7, I14, I21 – irradiated (1 kGy) samples of day 0,

7, 14 and 21)

216

Figure 45. PCA of bound aroma of pumpkin (C0, C7, C14, C21 – control samples

of day 0, 7, 14 and 21; I0, I7, I14, I21 – irradiated (1 kGy) samples of

day 0, 7, 14 and 21)

217

Figure 46. Effect of radiation treatment (2 kGy) and storage on (A) Conductivity

(B) Absrbance at 250, 280 and 320 nm (C) Browning index (absorbance

at 420 nm) of RTC ash gourd

220

Figure 47. Scanning electron micrographs of RTC ash gourd (A) Non-irradiated

control 0 d, (B) Radiation treated (2 kGy, 12 d) and c) Non-irradiated

control 12 d.

222

Figure 48. Effect of radiation processing (2 kGy) and storage on (A) PAL activity

and (B) PPO activity

225

Figure 49. (A) Effect of resorcylic acid (0, 50, 125 and 250 mM) on the browning

reaction rate of catechol. (B) Lineweaver-Burk plot of resorcylic acid

inhibition on the catechol-ash gourd PPO system

229

Figure 50. Plots for determining various kinetic parameters. (A) Plot of 1/Vmax vs

[I]; (B) Plot of Km/Vmax vs [I]; (C) Plot of V0/(V0-Vi) vs 1/[I]; (D)

Inhibitory effect of different concentrations of α-resorcylic acid on the

oxidation of catechol by ash gourd PPO

230

Figure 51. IR and NMR spectra of acetoin-3-O-β-D glucoside 235

Figure 52. A) Effect of varying concentration (0.178 – 4.4 µg/mL) of total aqueous

extract (Aq extract) of ash gourd, acetoin glucoside and acetoin on

tobacco leaf disc diameter on day 14 B) Effect of the various treatments

at the optimum concentration (0.44 µg/mL) on the diameter of leaf discs

during different time intervals

236

xxv

Figure 53. A) Increased leaf disc diameter for acetoin glucoside (0.44 µg/mL)

treatment on day 14. B) Germination in pearl millet seeds after treatment

with acetoin and its glucoside

238

Figure 54. Two dimensional gel from protein preparations of control and acetoin

glucoside treated tobacco leaves; Xt- protein over expressed in treated

sample, Xc – corresponding protein in control sample

239

Figure 55. Mass spectrum obtained by MALDI-ToF analysis of the protein (Xt)

over-expressed in response to acetoin glucoside treatment

240

Figure 56. RP-HPLC profile of ash gourd aq extract 244

Figure 57. Chromatogram obtained from GPC 246

Figure 58. Mass spectra of the identified alanine and valine 248

Figure 59. TLC separation of methanolic extracts obtained from control and

irradiated ash gourd stored for various storage periods

251

Figure 60. TLC profiles of unhydrolyzed (spot 1) and hydrolyzed (spot 2) extracts

with different solvent systems

252

Figure 61. HPLC chromatograms obtained at 254 and 237 nm for identification of

cucurbitacins

253

Figure 62. GC/MS profile obtained for hydrolyzate of the total extract 254

Figure 63. Chemical structure of the cucurbitacins identified 255

Figure 64. HPLC chromatograms obtained for drumstick aqueous extract and after

hydrolysis with myrosinase and sulfatase enzyme

258

Figure 65. GC/MS profile of hydrolyzed aqueous extract of drumstick 260

Figure 66. Chemical structure and mass spectra of isopropyl, 2-butyl and isobutyl

ITC

260

Figure 67. Structures of glucosinolates previously reported in Moringa species 261

Figure 68. HPLC chromatograms obtained from ash gourd, drumstick and pumpkin

at 280 nm

264

Figure 69. HPLC chromatograms obtained for control and radiation processed

pumpkin (0d and 21 d) carotenoids (450 nm)

270

xxvi

LIST OF TABLES

Table Title Page No.

Table 1. Richest vegetable sources of specific compounds 3

Table 2. Sensory descriptors reported for simple phenols 20

Table 3. The subgroups of flavonoids 21

Table 4. Phenolic acids isolated from vegetables 22

Table 5. Glucosinolates in different food sources 24

Table 6. Sensory scales used in the evaluation of food quality 29

Table 7. Instrumental methods for determination of vegetables quality 30

Table 8. Various applications of food irradiation 45

Table 9. Food items approved for radiation processing in India under PFA

rules

47

Table 10. Proximate composition of immature and mature ash gourd fruit 53

Table 11. Vitamins and minerals profile of mature ash gourd fruit 54

Table 12. Some common medicinal and pharmacological properties of

different parts of ash gourd

55

Table 13. Proximate composition and minerals and vitamins in drumstick

pods

58

Table 14. Medicinal and pharmacological activities of different parts of

drumstick tree

59

Table 15. Proximate and minerals and vitamins content in pumpkin pulp 63

xxvii

Table 16. Medicinal and pharmacological activities of pumpkin 64

Table 17. Experimental design for RTC ash gourd 112

Table 18. Experimental design for RTC drumstick 113

Table 19. Experimental design for RTC pumpkin 115

Table 20. Significance statistics, p values and signal to noise ratio (S/N) of

predicted models for ash gourd

116


predicted models for drumstick

117


predicted models for pumpkin

117

Table 23. Coefficients of the fitted polynomial representing the relationship

between the response and the process variable and R2 values for

RTC ash gourd

118



RTC drumstick

118



RTC pumpkin

119

Table 26. Criteria for various factors and responses for process optimization

and corresponding optimized solutions obtained

134

xxviii

Table 27. Quantitative distribution of volatile oil components identified in

isolates obtained from SDE, HVD and SPME

156

Table 28. Quantitative distribution of free volatile components obtained from

glycosidic precursors extracted by XAD and SPE

162

Table 29. ODP analysis of ash gourd SDE oil 167


SDE and SPME

172

Table 31. Quantitative distribution of free volatile components obtained from

glycosidic precursors extracted by XAD and SPE

177

Table 32. ODP analysis of drumstick SDE oil 183

Table 33. Bands isolated from TLC aromagram of drumstick oil with their

odor and major compounds identified

187


SDE and SPME

189


XAD and SPE isolates

196

Table 36. ODP analysis of pumpkin aroma (SDE) 201

Table 37. Effect of radiation processing and storage on POD activity of ash

gourd

225

Table 38. Quantitative distribution of different phenolic constituents in ash

gourd

227

Table 39. Correlation coefficients calculated between various factors 228

xxix

contributing in browning of RTC ash gourd

Table 40. The peaks collected from HPLC for preliminary screening of the

fractions possessing ACE inhibition activity

245

Table 41. Fractions collected from re-chromatography of region 2-4 min

obtained from preliminary HPLC analysis

245

Table 42. Fractions collected from GPC 246

Table 43. Peak area distribution of TLC spots as measured by TLC-

densitometry

251

Table 44. Rf values, UV absorption and the mass fragments of Cu C and Cu

E

255

Table 45. Major compounds detected in the collected peaks as identified by

GC/MS

259

Table 46. Quantitative distribution of major isothiocyanates in control and

radiation processed RTC drumstick at different storage periods

262

Table 47. Quantitative distribution of identified phenolic constituents in

drumstick

267

Table 48. Quantitative distribution of identified phenolic constituents and

carotenoids in pumpkin

268

CHAPTER 1

INTRODUCTION

2

1.1. Importance of vegetables in the diet

Vegetables form an important part of our daily diet. They provide nutrients vital for

health and maintenance of our body. Most vegetables are low in fat and calories with

none having cholesterol. They are important sources of both macro nutrients such as

fiber, carbohydrates, and micro-nutrients like potassium, folate (folic acid), vitamin A,

and vitamin C. Many of the colored vegetables, in particular, dark-green leafy,

cruciferous and deep-yellow-orange vegetables, are rich in vitamin C, carotenoids, folates

and a range of bioactive phytonutrients. A balance of the micro- and macro-constituents,

however, is more likely to be responsible for their health benefits than any single

compound. Several biologically active phytochemicals have been identified in food

plants. Table 1 lists vegetables that are rich source of specific compounds. However, apart

from a few exceptions, these compounds are also present in varying amounts in most

other fruits and vegetables.

In 1990, The World Health Organization (WHO) recommended a goal of at least 400 g of

vegetables and fruits daily (in addition to potatoes) including at least 30 g of legumes,

nuts and seeds [1]. This report, together with other reports from expert bodies, has been

translated into a recommendation for the consumption of at least five portions of fruits

and vegetables per day. The World Cancer Research Fund and American Institute for

Cancer Research have recommended that diets should be based primarily on foods of

plant origin, provided that such diets are nutritionally adequate and varied [2].

3

Table 1. Richest vegetable sources of specific compounds

There are evidences that diets rich in vegetables can decrease the levels of chronic

diseases. Extensive reports on the preventive effects of vegetables and fruits against

cancer are available in literature [3, 4]. In addition, new scientific evidence is emerging

supporting a protective role for vegetables in prevention of cardio-vascular diseases

(CVD), cataract formation, age-related macular degeneration, chronic obstructive

pulmonary disease, digestive disorders, and possibly hypertension. There may be several

biologically plausible reasons why the consumption of vegetables might slow, or prevent,

4

the onset of chronic diseases. Some of the phytochemical constituents present have

capacity to modify antioxidant pathways, detoxification enzymes, the immune system,

cholesterol and steroid hormone concentrations and blood pressure by acting as

antioxidant, antiviral and antibacterial agents.

Antioxidant effects of vegetables have been of recent interest. Dietary antioxidants have

been demonstrated to play a major health protective role. The huge literature data

currently available have shown that fresh fruits and vegetables possess high antioxidant

properties that are beneficial to human health [5, 6]. The oxidative damage to bio-

molecules is held responsible for CVD, cancer initiation, cataract formation,

inflammatory disease and several neurological disorders. The antioxidants are recognized

as bioactive compounds that protect bio-molecules from oxidative damage. Several trace

elements, such as manganese, copper, zinc, iron and selenium, are essential constituents

of the antioxidant metallo-enzymes: superoxide dismutase, glutathione peroxidase and

catalase. Vitamins C, E and the carotenoids & polyphenols, have received most attention

with respect to their antioxidant capability. They can interrupt free radical initiated chain

reactions of oxidation, or scavenge free radicals before they damage cellular components.

The in vivo antioxidant effects of different groups of compounds, have been studied, but

their metabolism is complex and effects in in vivo may be different both in extent and

mode of action from those observed in vitro model systems. Indeed, the health-promoting

effects of many phytochemicals are attributed mainly to their antioxidant activity,

although there could also be other modes. In a nutshell, vegetables are an important part

5

of our diet, which provide many beneficial nutrients, and protect against several chronic

diseases, apart from adding a specific flavor to the cuisine.

1.2. Minimal processing of vegetables

In the past few years, worldwide demand for convenience foods such as minimally

processed fresh cut fruits and vegetables has increased considerably. Fresh-cut products

are highly popular in Europe and in recent years have gained acceptance in Asia as well.

Changing lifestyles and eating habits as well as preference for fresh, healthy and natural

is a strong marketing drive for such products. Processed Ready-to-eat (RTE) fresh fruits

or ready-to-cook (RTC) vegetables provide convenience without greatly changing their

fresh like properties and human health benefits, with a minimal time of preparation before

consumption [7].

According to “The International Fresh-Cut Produce Association”, fresh-cut produce has

been defined as trimmed, peeled, washed, and cut into 100% usable product that is

subsequently bagged or prepackaged to offer consumers high nutrition,

convenience, and value while still maintaining freshness [8].

The USDA and FDA defined “fresh” and “minimally- processed” fruits and vegetables

as: fresh-cut (pre-cut) products which have been freshly-cut, washed, packaged and

maintained with refrigeration. Fresh-cut products are in a raw state and even

though processed (physically altered from the original form), they remain in a fresh

state, ready to eat or cook, without thermal processing, or treatments with additives

or preservatives [8].

6

Commercial production of RTC vegetables include washing, sorting according to size,

peeling, cutting, packaging and finally storage at refrigerated temperatures. However,

these products have a very short shelf life. For most fresh-cut produce, shelf-life is best

defined as the period within which the product retains acceptable quality for sale to the

consumer. It is therefore necessary to identify what ‘acceptable quality’ means before it

can be decided at what point the product no longer satisfies those expectations. From the

quality standpoint, it is desirable to preserve the characteristics of fresh-cut vegetables at

their peak. The most appealing attributes of these products include their fresh-like

appearance, taste and flavor, in addition to convenience without the use of preservatives

[9]. Fresh-cut processing increases respiration rates and causes major tissue disruption as

enzymes and substrates, normally sequestered within the vacuole, become mixed with

other cytoplasmic and nucleic substrates and enzymes. Processing also increases wound-

induced ethylene, water activity, and surface area per unit volume, which may accelerate

water loss and enhance microbial growth since sugars also become readily available [10].

These physiological changes may be accompanied by flavor loss, cut surface

discoloration, color loss, decay, increased rate of vitamin loss, rapid softening, shrinkage,

and a shorter storage life. Increased water activity and mixing of intracellular and

intercellular enzymes and substrates may also contribute to flavor and texture loss during

and after processing. Therefore, proper temperature management during product

preparation and refrigeration throughout distribution and marketing are essential for

maintenance of quality.

7

The critical factors for deciding the shelf-life of a fresh-cut produce can be classified into

the following categories: microbial spoilage, appearance and color, texture, flavor (taste

and aroma) and nutritional value.

1.2.1. Microbial spoilage

During processing of minimally processed vegetables such as peeling, cutting and

shredding, the surface of the produce is exposed to contamination with bacteria, yeasts

and moulds. Besides, the plant cellular fluids released during processing favor the growth

of micro-organisms by providing a nutritive medium for growth. Processing also

increases water activity and surface area per unit volume which may accelerate water loss

and enhance microbial growth [10]. Most of the minimally processed vegetables have low

acid range pH (5.8–6.0), high humidity and a large surface area which can provide ideal

conditions for the growth of microorganisms [11]. Thus, fresh cut vegetables have high

levels of microorganisms and several outbreaks of food-borne illnesses have been found

to be associated with these products [12]. In general, total counts of microbial populations

on minimally processed vegetables after processing range from 3.0 to 6.0 log cfu/g [13].

A number of micro-organisms have been found in fresh-cut products including

mesophilic microflora, lactic acid bacteria, coliforms, fecal coliforms, yeasts, and

pectinolytic microflora [13]. Some of these micro-organisms produce pectinolytic

enzymes which degrade the cell structure and as such provide more nutrients for

microbiological activity.

Physiological ageing of commodities could also increase microbial counts [14]. Moulds

are less important in minimally processed vegetables due to the intrinsic properties such

8

as slightly acid to neutral pH favoring bacteria and yeasts which generally overgrow

moulds [15]. Microorganisms impact the economic value of fresh-cut products by

decreasing product shelf-life, through spoilage, and by posing a risk to public health by

causing foodborne disease [13].

Mechanical wounding of vegetables enhances a diverse array of enzymatic pathways,

associated in many cases with generation of volatiles [16]. Increased concentration of

ethanol, acetaldehyde and some aliphatic alcohols such as 2-methyl-1-butanol, 3-methyl-

1-butanol, propanol has been reported to be produced by micro-organisms during storage

in various fresh-cut produce [17]. However, research on changes in volatile and non-

volatile compounds in minimally processed vegetables during storage in relation to

microbiological activity is scarce.

1.2.2. Appearance and color

Appearance is determined by physical factors including the size, the shape, the

wholeness, the presence of defects (blemishes, bruises, spots etc.), finish or gloss, and

consistency. Fruit or vegetable gloss are related to the ability of a surface to reflect light

and freshly harvested products are often more glossy [18]. Fresh-cut vegetable products

must appear to be fresh, generally indicated by the brightness of color and the absence of

visual defects or drip.

Color is derived from natural pigments present in fruits and vegetables. The primary

pigments include fat soluble chlorophylls (green) and carotenoids (yellow, orange and

red) and the water soluble anthocyanins (red, blue), flavonoids (yellow), and betalins

(red). Bright colors of fresh produce attract the buyers. However, color that is not

9

appropriate for the item, indicative of loss of freshness or lack of ripeness, can decrease

the consumer acceptance for the product. For example, white blush in cut carrots is a

quality defect [19]. Yellowing in green vegetables due to loss of chlorophyll is

unacceptable [20]. Wilting, browning, dull colors, and drip are all indicators of loss of

freshness in fresh-cut vegetables [21].

Browning is a serious quality defect in fresh-cut produce. Enzymatic and non-enzymatic

reactions may result in the formation of brown, gray, and black colored pigments. The

enzymes involved in browning reactions include phenylalanine ammonia lyase (PAL), a

key enzyme in the phenolic biosynthesis and polyphenol oxidase (PPO) as well as

peroxidase (POD), which catalyse the oxidation of polyphenolic compounds. In intact

plant cells, phenolic compounds in cell vacuoles are spatially apart from the oxidizing

enzymes present in the cytoplasm. Once tissues are damaged by processing such as

peeling and cutting, the mixing of the enzymes and phenolic compounds as well as the

easy oxygen diffusion to the inner tissues result in a browning reaction. Further, as a

result of minimal processing, phenolic content increases via wound-induced enhancement

in PAL (PAL; EC 4.3.1.5) expression. Oxidation of the phenols thus formed to quinones

catalyzed by PPO (PPO; EC 1.10.3.1) and peroxidase (POD; EC 1.11.1.7) and subsequent

polymerization of the quinones to relatively insoluble brown polymers (melanins) results

in unacceptable product [22]. It also leads to off flavors and losses in nutritional quality.

For example, russet (brown) spotting and brown stain [23] are undesirable visual defects

in lettuce. The main reactions as catalyzed by PPO are shown below (Figure 1). It has

been proposed that increase in PAL activity could be used as a predictive index of shelf

10

life [24]. An increased PAL activity has also been correlated with a decrease in shelf-life

and overall visual quality of minimally processed lettuce [24]. Increased enzymatic

activities have been reported in fresh-cut potato strips [25], broccoli florets (Brassica

oleracea var. italica; [26], and lettuce leaf segments [27]. Such visual defects decrease the

consumer acceptability and therefore marketability of the fresh-cut produce.

Figure 1. Reactions depicting mechanism of PPO action in browning development.

1.2.3. Texture

According to Bourne [28], the textural properties of a food are the “group of physical

characteristics that arise from the structural elements of the food, sensed by the feeling of

touch, are related to the deformation, disintegration and flow of the food under a force,

and measured objectively by functions of mass, time, and distance”. Textural parameters

of fruits and vegetables are perceived with the sense of touch, either when the product is

picked up by hand or chewed. Consumers have clear expectations regarding the texture of

fresh-cut vegetables. Salad vegetables like lettuce, carrot, celery, and radish should be

crisp. Undesirable textural attributes are the opposite of the desirable ones. For example,

wilted lettuce, limp carrots or celery, and flaccid radish are unacceptable.

11

Texture is derived from turgor pressure, and the composition of individual cell walls. Cell

walls are composed of cellulose, hemicelluloses, pectic substances, proteins, and also

lignins in the case of vegetables. In processed fruits and vegetables, changes in texture are

strongly related to transformations in cell wall polymers due to enzymatic and non-

enzymatic reactions. Cellulose and hemicellulose show minimal changes in structure and

composition in most plant based foods [29]. Most of the changes observed in plant based

foods are ascribed to transformations in pectin structure and composition. These changes

are strongly influenced by the processing steps and conditions. Apart from mechanical

injury imposed by processing operations, microbial growth also bring textural changes in

minimally processed vegetables during storage [13]. The rapid texture breakdown

observed in cut vegetables during storage is often the result of higher aerobic

psychotrophic counts. Different micro-organisms produce pectinolytic enzymes including

pectate lyase, polygalacturonase and pectin methyl esterases resulting in textural changes.

The most commonly isolated pectinolytic bacterial species are Erwinia and

Pseudomonas. Pectinolytic yeasts and moulds include Trichosporon sp and Mucor sp,

respectively [13].

While generally flavor is being cited as the most important quality attribute, textural

defects and the interaction of flavor and texture are more likely to cause rejection of a

fresh product [30]. Consumer and taste panel responses indicate that individuals are

actually more sensitive to small differences in texture than flavor [8], making texture a

crucial parameter for acceptability.

12

1.2.4. Flavor (taste and aroma)

An overall flavor of food results from the combined effect of its various constituents on

the human olfactory organs. Taste sensation is perceived once the food product is taken in

the mouth, while smell (olfaction) can be sensed by the odor of an object at a distance.

1.2.4.1. Taste

Taste can be classified into five basic categories - sweetness, sourness, saltiness,

bitterness and umami. In Asian countries within the sphere of mainly Chinese and Indian

cultural influence, pungency (piquancy or hotness) had traditionally been considered a

sixth basic taste. Sweetness, usually regarded as a pleasurable sensation, is produced by

the presence of sugars, some proteins, and a few other substances. It is often connected to

aldehydes and ketones, which contain a carbonyl group. Sourness in the taste is detected

by the acidity content of the food product. The sourness of substances is rated relative to

dilute hydrochloric acid, which has a sourness index of 1. Citric acid, malic acid and

oxalic acid are the acidic compounds attributing sourness to fruits and vegetables. Many

of the fruits are naturally sour in taste such as lemon, grape, orange, and tamarind.

Saltiness is a taste produced primarily by the presence of sodium ions. The saltiness of

substances is rated relative to sodium chloride (NaCl), which has an index of 1 [31].

Bitterness is an undesirable taste found in some fresh-cut vegetables such as salad greens

[32] and vegetables of cruciferae family. When Cruciferae cells are ruptured,

glucosinolates undergo enzymatic hydrolysis with the endogenous myrosinase enzymes,

releasing thiocyanates, isothiocyanates [33], sulphate, and glucose [34]. These breakdown

products cause bitter taste. Umami is an appetitive taste and is described as a savory [36]

13

or meaty [36]. It can be tasted in cheese and soy sauce, and while also found in many

other fermented and aged foods, this taste is also present in tomatoes, grains, and

beans[36]. The amino acid, glutamic acid is responsible for umami taste [37] but some

nucleotides (inosinic acid and guanylic acid [37] can act as complements, enhancing the

taste. Processing and packaging precautions must be taken to ensure that off-odors and

off-flavors do not jeopardize the marketability of the fresh-cut vegetables.

1.2.4.2. Aroma constituents

Although taste sensations are very important, it is the presence of trace amounts of

(usually) many volatile compounds which are crucial in determining the flavor quality of

a food product.

The aroma of vegetables is generally released during processing such as cutting and

cooking. It is generated due to the presence of a large number of organic volatile

compounds which are present in extremely small concentrations. Two largest groups that

contribute to natural odors are the aliphatics derived mainly from fatty acids and

terpenoids synthesized by the maevalonate or the methylerythritol pathway. Among the

aliphatic compounds derived from fatty acids with carbon chains between two to

seventeen, the C6 compounds such as (Z)-3-hexenyl acetate, hexenol, hexenal and

hexanol belong to a well known group of green-leaf volatiles (GLVs) found in several

fruits and vegetables. Terpenes constitute the largest family of natural plant products

including fruits and vegetables. They are made up of homologous series of repetitive five

carbon isoprene units in their structure. These include the monoterpenes (C10, 2 isoprene

units), sesquiterpenes (C15, 3 isoprene units), diterpenes (C20, 4 isoprene units),

14

triterpenes (C30, 6 isoprene units), tetraterpenes (C40, 8 isoprene units) and polyterpenes

([C5]n, where n may be 9-30,000). Among these, the monoterpenes and sesquiterpenes

are the major constituents of several essential oils derived from plants and plant products.

Terpenes can be further sub divided into terpene hydrocarbons and oxygenated terpenes

depending on the nature of their functional groups. Some of the monoterpene

hydrocarbons such as myrcene, α-pinene and sabinene are widely distributed in fruits and

vegetables and have pleasant and characteristic odor. Oxygenated terpenes commonly

exist as alcohols, aldehydes, ketones and esters.

Other odorant chemical classes include the benzenoids and phenylpropanoids derived via

the phenylpropanoid pathway, lactones derived from hydroxyl fatty acids and C-5

branched chain compounds derived from branched-chain fatty acids. Among the nitrogen

containing compounds, amines, oximes and indoles are the most common. Sulfur

containing compounds such as hydrogen sulfide, methanethiol, dimethyl sulfide and

isothiocyanates are derived from amino acid metabolism. Figure 2 represents some

representative examples of aroma compounds existing in the nature.

In vegetables, the volatiles representing their characteristic flavor are generally esters,

aldehydes, alcohols, terpenes or their derivatives. Sometimes, a single compound alone

can approximate the flavor of a product and thus is termed as “impact compound”.

However, in other cases a combination of several constituents that, together, interact with

the receptors from the nasal mucosa creates a sensory impression in the brain typical of

the product [38].

15

Figure 2. Representative aroma compounds of vegetables

Besides the volatile constituents that contribute to odor, there exists a class of non-volatile

glycosidically bound odor precursors that are widely distributed in the plant kingdom

[39]. Although odorless, they are able to release free aroma by enzymatic hydrolysis

during processing such as cutting and cooking and hence are considered to be potential

aroma compounds that play a crucial role in the overall food quality. Some of the bound

volatile compounds have unique odor properties that provide the characteristic flavor to a

food product. The aglycone bound to the glycoside moiety may include both terpenoid

and non-terpenoid structures. Mono and sesquiterpenoids, aliphatic alcohols, alkyl

phenols and norisoprenoids are the most prominent. Generally the sugar which is directly

bound to the aglycone is β-D-glucose. This glucose moiety may or may not be further

substituted with additional sugar units. The second sugar unit is reported to be either α-L-

16

arabinofuranose, α-L-rhamnopyranose, β-D-xylopyranose, β-D-apiofuranose, or β-D -

glucose (Figure 3). Most of the hydrolases employed for release of volatile aglycones are

exo-glycosidases. Initial step in the cleavage of disaccharidic conjugate therefore require

the action of α-L-arabinosidase, α-rhamnosidase, β-D-xylosidase, or β-D-apiosidase, for

the cleavage of the intermediate sugar linkage. A second hydrolytic step further liberates

the aglycone moiety by β-glucosidase activity [40]. Only in the case of gentiobiosides (β-

D-glucopyranosyl-(1-6)- β-D-glucopyranosides), where the disaccharidic sugar consists

of two glucose units, β-D-glucosidase activity is able to liberate the aglycone in a two-

step mechanism [41].

Figure 3. Structrue of glycosidic portion of aroma glycosides

The intact fresh vegetables are nearly odorless and are indistinguishable from each other.

However, processing such as cutting and cooking, results in liberation of characteristic

17

aroma from their precursors. Very few reports however exist on the nature of free as well

as bound aroma compounds in vegetables, especially in Indian vegetables. Further,

reports on the effect of various processing on aroma of vegetables and their products are

lacking. Therefore, it is of interest to study the volatile profile of vegetables and to

identify the role of these compounds in providing characteristic aroma to these products.

1.2.5. Nutritional value

The beneficial effects of vegetables have been attributed to non-essential food

constituents, which are known as phytochemicals or bioactive compounds. These

phytochemicals are considered to be biologically active secondary metabolites that in

many cases provide color and flavor, and are commonly referred to as phytoprotectants or

nutraceuticals [42]. Plant derived phytochemicals have been shown to be associated with

many health-promoting effects such as protection against inflammation, cardiovascular

diseases, diabetes, asthma and cancer [43]. Fresh vegetables are good sources of dietary

fiber, minerals, vitamins, and other beneficial phytochemicals. The major classes of

phytochemicals important in vegetables include carotenoids, phenolics, and

glucosinolates.

1.2.5.1. Carotenoids

Fruits and vegetables contain different amounts and types of carotenoid [44]. Chemically

carotenoids are polyisoprenoid compounds and can be classified into two main groups:

(a) carotenes composed only of carbon and hydrogen atoms and (b) oxygenated

derivatives, xanthophylls, that contain at least one oxygen function such as hydroxy, keto,

epoxy, methoxy or carboxylic acid groups. Their structural characteristic is a conjugated

18

double bond system which influences their chemical, biochemical and physical

properties. This class of natural pigments occurs widely in nature. They are responsible

for the attractive colors of many flowers, fruits and vegetables [44]. This attribute is of

great importance in foods, since color is often a criterion of quality and is typically

modified by food processing [45]. Carotenoid content in fruits and vegetables depends on

several factors such as genetic variety, maturity, postharvest storage, processing and

preparation.

The physiological role of these compounds has resulted in a great interest in their

biological function [46]. In addition to the provitamin A activity of some carotenoids,

they also have other functions, such as antioxidants and enhancers of the immune

response. Furthermore, some of them are involved in the cell communication.

Xanthophylls have also been shown to be effective as free radical scavengers [47]. Figure

4 shows some representative examples of the carotenoids found in fruits and vegetables.

Figure 4. Some representative carotenoids found in vegetables.

19

1.2.5.2. Phenolics

Phenolic compounds refer to the main classes of secondary metabolites in plants.

Chemically, phenols are cyclic benzene compounds possessing one or more hydroxyl

groups associated directly with the ring structure. Phenolic compounds are important in

deciding overall quality since they contribute to organoleptic characteristics such as

colour, astringency, bitterness, and aroma. Table 2 shows the organoleptic notes

associated with certain phenolic compounds. Phenolics are produced in plants as

secondary metabolites via the shikimic acid pathway [48]. Phenylalanine ammonialyase

(PAL) is the key enzyme catalyzing the biosynthesis of phenolics from the aromatic

amino acid, phenylalanine [48]. They can be classified based on the number and

arrangement of the carbon atoms as flavonoids (flavonols, flavones, flavan-3-ols,

anthocyanidins, flavanones, isoflavones and others) and nonflavonoids (phenolic acids,

hydroxycinnamates, stilbenes and others). They are commonly found conjugated to

sugars and organic acids.

1.2.5.2.1. Flavonoids

Flavonoids are polyphenolic compounds comprising fifteen carbons with two aromatic

rings connected by a three-carbon bridge (C6-C3-C6). They are the most abundant

phenolic compounds found throughout the plant kingdom. The individual flavonoid

subgroups are shown in Table 3. Flavonols are the most widespread of the flavonoids.

Kampferol, quercetin, isorhamnetin, myricetin and their O-glycosides are some of the

flavonols reported in vegetables. In fresh vegetables, generally flavonols exist only as

glycosidic precursors or acylated by various hydroxycinnamic acids [49]. Within the

20

flavonoids, anthocyanins are the most important group of water-soluble colored plant

pigments, possessing antioxidant activity and other useful biological properties. They are

generally found in the form of glycosides, with aglycones being rarely found. They are

involved in protecting the plants against excessive light and also have an important role in

attracting pollinating insects. The stability, color intensity and potential biological activity

of anthocyanins is determined by their chemical structure.

Table 2. Sensory descriptors reported for simple phenols

21

Table 3. The subgroups of flavonoids (Table adapted from Karakaya, S. (2004) [50])

22

1.2.5.2.2. Phenolic acids

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the

plant kingdom. Hydroxycinnamic acids such as ferulic, sinapic, caffeic, and p-coumaric,

are among the most widely distributed in plants. Table 4 shows the phenolic acids

isolated from various vegetables [51]. They occur in most tissues in a variety of

conjugated forms. Esters and amides are the most frequently reported types of conjugates,

while glycosides rarely occur. The most abundant member of dietary hydroxycinnamates

are ferulic and caffeic acids [52]. Most vegetables contain caffeic, ferulic, sinapic or

coumaric acid conjugated with quinic acid and/or esterified with sugars [53].

Table 4. Phenolic acids isolated from vegetables.

23

Phenolic acids have been known to possess sensory properties described as being

astringent and bitter that are not considered very desirable in some products.

1.2.5.3. Glucosinolates

Glucosinolates (GSLs) constitute a well defined group of specialized plant metabolites.

They are predominantly found in crucifers. Several other plant families in the same plant

order as the Brasicaceae, the Capparales (e.g., the Capparidaceae, Moringaceae,

Resedaceae, Stegnospermaceae, and Tovariaceae) have been found to possess

glucosinolates. Table 5 represents glucosinolates identified in various food products.

A large body of epidemiological evidence indicates that the chemoprotective effects of

Brassica vegetables against initiation of tumors caused by chemical carcinogens may be

due to glucosinolates and their degradation products [54]. There is a considerable interest

in recent years in optimizing GSL content and composition for plant protection and

human health. GSLs are also responsible for the bitter acidic flavors of Brassicacea

species. Their hydrolytic by-products such as isothiocyanates, nitriles, and thiocyanates

are responsible for the hot and pungent taste.

Structurally glucosinolates are anions composed of thiohydroxymates carrying an S-

linked β-glucopyranosyl residue and an O-linked sulfate residue, and with an amino acid

derived, variable side chain (Figure 5). Based on the nature of side chain, they are broadly

classified as alkyl, aromatic, benzoate, indole, multiple glycosylated and sulfur containing

side chains. The enzyme myrosinase (β-thioglucosidase glucohydrolase; EC 3.2.3.1)-

activated in damaged plant tissue and also present in the microflora of the human

digestive tract converts these glucosinolates to a number of compounds including

24

thiocyanates, nitriles and isothiocyanates (Figure 5). These hydrolysis products, many of

which possessing biological activity, vary depending on the plant species, side-chain

substitution, cell pH, and cell iron concentration (55, 56].

Table 5. Glucosinolates in different food sources.

25

Figure 5. General structure of glucosinolates and their enzymatic degradation products.

(Adapted from Rask et al., 2000 [57]).

In contrast to intact GLS, hydrolysis products are responsible for the characteristic aroma

of Brassicaceae plants and have been of interest for their diverse biological activities,

ranging from antimicrobial, antioxidant and anticancer activities [58]. Many of these have

biocidal activity against a wide variety of organisms such as insects, plants, fungi,

bacteria and human health effects [59]. For example, sulforaphane [1-isothiocyanato-4-

(methylsulfinyl)butane], a degradation product of the glucosinolate glucoraphanin [4-

(methylsulfinyl)butyl glucosinolate], is a potent inducer of phase II detoxication enzymes,

that are strongly correlated with the prevention of certain types of cancer [60].

1.2.5.4. Triterpenoids

Triterpenes or triterpenoids are terpenes consisting of six isoprene units and having

molecular formula C30H48. Triterpenoids are biosynthesized in plants by the cyclization of

squalene, a triterpene hydrocarbon and precursor of all steroids [61]. They are used for

26

anti-inflammatory, analgesic, antipyretic, hepatoprotective, cardio tonic, sedative and

tonic effects [61]. They also demonstrate antitumor efficacy in preclinical animal models

of cancer [61]. They can further be sub classified into diverse groups including

cucurbitanes, cycloartanes, dammaranes, euphanes, friedelanes, holostanes, hopanes,

isomalabaricanes, lanostanes, limonoids, lupanes, oleananes, protostanes, sqalenes,

tirucallanes, ursanes and miscellaneous compounds [61]. Among them, cucurbitacins are

well-known for their cytotoxic behavior and broad range of bioactivities such as

antitumor, anti inflammatory, antimicrobial, antihelminthic, and cardiovascular properties

[62]. Cucurbitacins are oxygen containing compounds possessing double bonds and

derived from cucurbitanes (C30H54) that have a triterpene hydrocarbon skeleton (Figure

6). They usually occur as β-2-monoglycosides, the sugar moiety being D-glucose or L-

rhamnose. They possess the biogenetically unusual 10acucurbit-5-ene-[19(10-19b)-abeo-

10a-lanostane] skeleton [63], which has mainly been reported in the Cucurbitaceae but is

also known to occur in other plant families [63].

Figure 6. Chemical structure of cucurbitane (C30H54).

27

1.3. Quality measurement of fresh-cut produce - Sensory and instrumental

measurements

The quality of fresh-cut produce can be measured by sensory and instrumental methods.

In general, sensory methods are more useful in developing new products and determining

product standards while instrumental methods are superior in measuring quality on a

routine basis [21].

1.3.1. Sensory methods of quality measurement

Sensory methods are very important for quality measurement of fresh-cut produce. Since

human perception is involved in sensory testing, quality attributes are clearly defined in

terms that are relevant to consumer acceptability. The sensory panels need to be

extensively trained as highly variable results can be produced if training is inadequate.

Trained sensory panels can be used to identify small differences in the samples processed

with different treatments (Difference tests) or for descriptive analysis of a product.

Descriptive analysis involves the development of a lexicon [64] which is the list of terms

used as descriptors with their precise definitions. Lexicons can be extensive with up to 50

descriptors or with as few as 5 descriptors. Selection of a lexicon is followed by training

of the panel to acclimatize them with the descriptors in order to ensure that the results are

accurate and precise.

After completion of the training, the evaluation of the samples is conducted in partitioned

booths using one of the several evaluation methods such as Spectrum [64] and

Quantitative Descriptive Analysis (QDA; [65]). The primary differences between the two

28

techniques are that Spectrum uses standards for the descriptors and involves more

extensive training than QDA. A more limited approach to descriptive analysis is the use

of an experienced panel. Experienced panelists have had some training on similar

descriptors in the past, but the panel is usually provided with a limited number of

descriptors for evaluation. The panel is convened with a few training sessions primarily to

ensure that the panelists are familiar with the terminology. Usually two to five judges

evaluate the samples independently of each other to prevent bias.

However, to determine the preference (which samples are preferred over others) of a food

product, large numbers of na¨ıve panelists are generally used [66]. The preference tests

give an idea about what consumers like and what they do not like.

The most frequently used evaluation scale is the 9-point hedonic scale [64]. Some more

useful scales include the 5-point willingness to purchase [67] and the 3-point acceptability

scale [68]. Table 6 represents the sensory scales generally employed in the food industry

for deciding the acceptability of a product.

However, the use of a single sensory technique provides limited information. Integration

of two or more techniques can be a powerful tool in the quality evaluation of fresh-cut

produce [69].

To summarize the main points, sensory analysis helps to determine which attributes are

important to the consumer. Difference tests can determine if individual units are

noticeably different, and sensory descriptive analysis can identify the attributes that cause

the differences. When carefully coordinated, the sensory tests can be very effective in

developing new products and establishing quality standards.

29

Table 6. Sensory scales used in the evaluation of food quality

1.3.2. Instrumental methods of quality measurement

Apart from sensory analysis which is a crucial step in product development, a wide range

of instrumental techniques for quality assessment of fresh-cut produce in terms of color,

appearance, flavor, texture, and nutritional quality are available. These techniques, also

termed as objective methods, are advantageous in a way that they tend to provide accurate

and precise results. The results of instrumental tests can generally be related directly to

chemical and physical properties allowing the investigator to gain a mechanistic

understanding of observed differences. Instruments tend to be more sensitive to small

differences between samples and may be able to detect trends in quality loss before they

can be detected by humans [70]. Large amounts of data can be generated by using

instruments without any fatigue or loss of sensitivity/ alertness towards analysis. This

30

attribute makes them excellent monitors in Quality Control operations. Instrumental

methods of measuring color, texture, aroma, and flavor in fruits and vegetables as

described by Kader [71] are listed in Table 7.

Table 7. Instrumental methods for determination of vegetables quality

1.3.2.1. Color

Color may be determined using nondestructive methods based on visual or physical

measurements. These methods are based on evaluation of either the light reflected from

the surface of a product or transmitted through it. There are three components necessary

for the perception of color - 1) a source of light, 2) an object that modifies light by

reflection or transmission and 3) the eye/brain combination of an observer. Easy and fast

31

methods such as use of simple color charts and dictionaries are routinely used in the field,

packing house, fresh-cut processor facility or retail store. They have the advantage of

requiring no specialized equipment, but may be standardized through the use of color

charts or discs. However, human differences in perception might give erroneous results.

Inadequate or poor quality of the available light may also affect accuracy [18]. On the

other hand, instrumental methods are less variable and can be used to measure small

differences; however, they may be slower than the sensory measurements.

Each pigment in vegetables corresponds to a primary hue—red, blue, and green [72].

The Commission Internationale de l’Eclairage (CIE) or International Commission on

Illumination is the international body that governs the measurement of color. Color space

may be divided into a three-dimensional (L, a and b) rectangular area (Figure 7) such that

L (lightness) axis that goes vertically from 0 (perfect black) to 100 (perfect white) in

reflectance or perfect clear in transmission [73]. The “a” axis (red to green) considers the

positive values as red and negative values as green; 0 being neutral. The “b” axis (blue to

yellow) expresses positive values as yellow and negative values as blue; 0 being neutral.

Figure 7. Diagram depicting three dimensional L, a and b color space

32

Color may be determined instrumentally using either colorimeters or spectrophotometers.

Colorimeters give measurements that can be correlated with human eye-brain perception,

and give tristimulus (L, a and b) values directly [73]. Colorimeters are typically quite

rugged and desirable for routine quality control measurements. Spectrophotometers

provide wavelength-by-wavelength spectral analysis of the reflecting and/or transmitting

properties of objects, and are more commonly used in research and development

laboratories [73].

Pigments of vegetables may also be analyzed quantitatively by extraction with specific

solvents, filtration, and the use of various methods based on spectrophotometry.

Separation using reversed phase high performance liquid chromatography (HPLC) may

be useful prior to measurement of absorption of light in the UV/visible wavelength

spectrum. Colorimetric methods are based on the Lambert-Beer law which describes the

relationship between the concentration of a substance and its color intensity:

E = ε’lc

The extinction coefficient E is proportional to ε’, the molar extinction coefficient of the

substance, l, the length of the light path in centimeters, and c, the concentration in g l−1

.

1.3.2.2. Texture

Most methods used for the evaluation of the textural properties of vegetables comprise a

wide range of simple and rapid tests, including puncture, compression, extrusion, shear,

and others. The puncture test, which is a force measuring method that has the dimensions

mass, length, and time, is the most frequently used method for textural evaluation. It

33

consists of measuring the force and/or deformation required to push a probe or a punch

into a food material to a depth that causes irreversible damage or failure. Puncture probes

of a specific diameter may be easily fitted to laboratory-scale instruments such as the

Maturometer, the Instron, and the Texture Technologies TAXT2 machine for more

controlled measurements [74].

1.3.2.3. Flavor- Aroma and Taste

Flavor may be evaluated with either instrumental or sensory methods, however, sensory

methods are the most critical to this particular attribute. Therefore, flavor may be the most

challenging quality attribute to both measure and correlate to consumer acceptability.

1.3.2.3.1. Analysis of aroma compounds

There are several challenges in the analysis of aroma compounds. It involves their

isolation from a given product, further separation, quantification and then correlating their

odor to the chemical structure. Odor molecules can be found in any class of chemical

compounds and thus can have widely varying structure and polarity. Further, these

compounds are labile and susceptible to chemical changes. For example, terpenes can

undergo photooxidation/ rearrangements in the presence of light or unsaturated

compounds can undergo polymerization in the presence of air. Such changes are of

concern during sample preparation as they generate artifacts. The problem is further

compounded by their occurrence in minute quantities (ppb-ppt) requiring sensitive and

sophisticated instruments for their detection and analysis. Contribution of an odorant to

the total aroma of a food product is additionally dependent on its threshold, which results

34

in even a compound present in minute amount having a greater contribution to the odor

than constituents present in very large concentration. Two terms namely "odor impact

compounds" and "contributory odor compounds" have thus been used to define top odor

notes. The former refers to those compounds that actually contribute to the typical odor of

a product while the latter modify the typical odor to a pleasant and acceptable note.

1.3.2.3.2. Isolation of aroma principles

Aroma isolation from a given matrix involves crushing, homogenizing, blending or

extracting the matrix with minimum loss in these constituents. Fresh materials contain

active enzymes in their tissues that can alter the aroma profile once cellular disruption has

occurred. The complex nature of the food matrix containing proteins, fats or

carbohydrates makes the process of aroma isolation complicated. Solvent extraction

using organic solvents at room or sub ambient temperatures is one of the most common

and conventional method for extraction of aroma compounds. The nature of the solvent

used, polar or non-polar, depends on the type of compounds to be isolated and identified.

Drawbacks of this method, however, are the co-extraction of non-volatile constituents

posing problems in recovery of volatile odors and the solvent impurities interfering in the

analysis, and falsely identified as a sample constituent, particularly when large amounts

of solvent are used. Supercritical fluids are now being used particularly in supercritical

fluid extraction, thus avoiding the problem posed by the use of organic solvents. SFE uses

variety of fluids (typically, CO2 possibly modified with very low amounts of organic

solvents as modifiers), high pressures (2000-4000 psi) and elevated temperatures (50-

35

150°C). The method produces clean extracts with no additional concentration for gas

chromatographic (GC) analysis.

Steam distillation techniques such as simultaneous distillation extraction (SDE) are

widely used for the isolation of aroma from vegetables. However, isolation of odor

molecules using SDE can bring about drastic changes in odor quality such as formation of

cooked odors. High vacuum low temperature distillation, solid phase micro-extraction,

microwave-assisted extraction, pressurized fluid extraction and head space techniques are

generally employed for extracting the fresh aroma of the vegetables to avoid artifact

formation due to thermal treatment in steam distillation techniques.

High vacuum distillation is a mild technique operated at or below the ambient

temperature wherein the potential loss of very low boiling volatiles is minimized. Artifact

formation observed in many other distillation techniques is eliminated. The method

provides for true odor of a given product. However, only low yield of aroma concentrates

are obtained compared to steam distillation technique and thus is generally used for the

isolation of labile compounds or when the aroma impact components have to be identified

or confirmed.

Microwave-assisted extraction involves extraction using microwave transparent solvents.

Contents of the cells broken by internal heating at microscopic level by microwave

energy come into contact with surrounding cool solvents avoiding thermal degradation of

labile odors. However, concentration of the isolate prior to GC analysis is required.

Headspace analysis is another very useful technique for rapid identification of volatile

aroma in headspace above a food sample. Two methods generally employed are the static

36

and dynamic headspace techniques. Static headspace involves enrichment of volatiles in a

closed chamber and is generally used when low volatile emitting samples are to be

investigated. The method has the disadvantage of long sampling times. In order to reduce

sampling time and to obtain better yields in static headspace, solid phase microextraction

(SPME) is currently employed for collection and further identification by GC. The

method is fast, effective, simple and is based on adsorption-desorption technique using an

inert fiber coated with different types of adsorbents varying in polarity and thickness and

used depending on the application demanded. Non-polar volatile compounds are

effectively extracted with non-polar fiber coatings such as polydimethylsiloxane (PDMS)

while polar volatiles can be extracted with PDMS/divinylbenzene or PDMS/Carboxene

polar fibers. The fibers equilibrated with headspace volatiles are directly transferred to the

GC injection port where the compounds are desorbed thermally. This eliminates solvent-

mediated desorption, thereby reducing the risk of solvent contamination. Some of the

limitations of SPME include (i) no repeated injections are possible, and (ii) the low

amount of material adsorbed on SPME fibers makes the method amenable only to GC

analysis but not for structure elucidation of unknown compounds. Consistent sampling

time, temperature and sample volume are crucial to obtain comparable results.

In dynamic headspace, a continuous air stream is passed over the headspace, and the

odorous compounds carried along with air are trapped on to porous polymeric traps such

as Tenax (2,6- diphenyl-p-phenylene oxide) and Porapack (ethylvinylbenzene-

divinylbenzene) or carbon-based adsorbents such as activated charcoal, carbon molecular

sieves and graphitized carbon black. The volatiles can be directly desorbed from the

37

adsorbent into the GC in case of porous polymers while in case of carbon-based

adsorbents the volatiles are generally eluted with small volumes (30-40 µl) of organic

solvents. Although, Tenax has a lower capacity than Porapack, it has higher thermal

stability and thus is particularly suited for thermal desorption of volatile compounds in

GC analysis. A combination of tenax and charcoal with high adsorbing capacity have

been employed for trapping full range of odorants varying widely in their polarities.

Dynamic headspace permits collection of large amount of volatile aiding in identification

of unknown structures.

1.3.2.3.3. Separation and detection of aroma compounds

As discussed earlier, a volatile odor extract is generally made up of an aroma bouquet

containing anywhere from a few to several different constituents present in varying

amounts. The individual components have to be separated from the mixture to facilitate

their identification. The most commonly used method of separation is the

chromatographic technique based on adsorption / partition of constituents between two

phases. Among the chromatographic techniques, gas liquid chromatography (GLC) is the

most efficient technique for the separation, identification and quantification of volatile

organic compounds. The separations are affected by both sample preparation and

isolation procedure. The nature of the stationary phase in the column has a distinct impact

on separation efficiency. Commonly used stationary phases are the non polar dimethyl

polysiloxanes (DB-l, DB-5, CPSil 5, SE-30 and OV-1) and the more polar polyethylene

glycol polymers (CarbowaxTM

20 M, DB-Wax and HP 20M). For different stationary

38

phases, retention index data such as kovats index system have been developed to facilitate

compound characterization and identification. Detection of peaks can be carried out using

two types of detector. First type include the flame ionization detector (FID) and the

thermal conductivity detector (TCD) that provide the retention times while the second

type include the mass spectrometer (MS) and the Fourier transform infrared (FT-IR)

spectrometers that aid in obtaining structural information. FID is a highly sensitive

detector (0.05 – 0.5 ng per compound) and is based on detection of ions formed when

organic compounds are burnt in a flame, while TCD, a less sensitive detector, operates by

differential thermal conductivity of gaseous mixture. MS with a sensitivity of 0.1 - 1 ng

per compound, relies on generation of positively charged molecules/and molecule

fragments from compounds separated on the GC column. Several comprehensive mass

spectral libraries (WILEY, NIST MS data base) have been established and are currently

used in EI-MS searches for tentative compound identification. Unambiguous

identification can, however, be achieved by comparison of mass spectrum with that of

authentic standards analyzed on the same column and determination of kovats indices on

at least two columns with different polarities. Multi dimensional GC, wherein

constituents of volatile isolates are separated by sequentially passing through two

successive columns, is used for separation of complex isolates such as essential oils.

Only a small fraction of the large number of volatiles in a given aroma isolate actually

contributes to the odor. The distinction between odor active compounds and the whole

range of volatiles present in an isolate from a food is thus an important task in odor

analysis. Sniffing the gas chromatographic effluents of an isolate in order to associate

39

odor activity with eluting compounds is an interesting approach. Principal analytical

technique used in this regard is the gas chromatography-olfactometry (GC-O) technique

that aids in assessing the aroma potential of a compound. GC-MS-O provides a means of

identification of compounds. First formal approach to determine contribution of a volatile

compound to the overall odor is based on its odor activity values (OAV) which is defined

as the ratio of concentration of the volatile compound to its odor threshold. The so-called

odor impact compounds can be distinguished from other volatiles by their high OAV.

Among the GC-O techniques, the dilution sniffing methods namely Aroma Extraction

Dilution Analysis (AEDA) and the Combined Hedonic Aroma Response measurements

(CHARM) are routinely used for identification of odor active constituents. Both methods

involve sequential dilution of samples. In AEDA, the most widely used method, each

dilution is sniffed after injection on to the GC until no significant odor is detected. The

result is then expressed as a flavor dilution (FD) factor which is the ratio of concentration

of the odorant in the initial extract to its concentration in the most dilute extract in which

an odor was detected. However, the method requires evaluation by a trained panel. The

methods thus lack reproducibility due to variability within and between sensory panelists.

A more recent method based on frequency of detection of a given odor, rather than

perceived intensity termed as Olfactory Global Analysis, eliminates the drawback of

dilution techniques. The repeatability of the method has been demonstrated to be

satisfactory despite the use of untrained sensory panel. The method is also twice as fast as

other GC-O techniques. A group of assessors is, however, a prerequisite for a reliable

40

GC-O analysis and thus the results can be systematically affected due to decreasing

alertness of the assessor.

1.3.2.3.4. Isolation and identification of bound aroma glycosides

The glycosidic aroma precursors are isolated from plant extracts (aqeuous or aqueous

methanol) by selective retention on C-18 reversed phase adsorbent [75] or amberlite

XAD-2 resin [76], followed by the desorption of the retained glycosides using EtOAc or

MeOH. The eluate so obtained is generally subjected to acidic or enzymatic hydrolysis

and the liberated aglycones are analyzed by GC/MS for identification. However, to

characterize intact glyco-conjugates, sophisticated steps of chromatographic separations

need to be followed. For preliminary separations, LC techniques are generally suited,

such as preparative HPLC and size exclusion chromatography. The isolates are then

subjected to repeated purification steps before identification. Purified compounds are

identified based on spectroscopic techniques such as NMR, IR, MS, or chiroptical

methods or analysis of hydrolysis products by GC/MS.

1.3.3. Analysis of bioactive phytochemicals

The phytochemicals may be extracted depending on their water or lipid solubility and are

typically analyzed using HPLC. Major anti-oxidant phytochemicals such as polyphenolic

compounds like phenolic acids, flavonols, flavones or the flavonoids, are extracted in

methanolic or ethanolic extract of the tissue, which after preliminary cleanup are

subjected to HPLC or LC/MS analysis for separation and identification of the compounds

[77]. In the case of colored phytochemicals like anthocyanins, it is possible to estimate

its content by measuring the intensity of color or a/b value with a colorimeter, but such a

41

physical method is not available for most nutrients. Glucosinolates generally require hot

aqueous alcohols such as methanol: water (70:30) for their isolation from plant materials

in order to prevent their hydrolysis by myrosinase [88]. A prior separation into groups

normally precedes their identification and quantification by HPLC-MSn. Presence of

sulfate groups facilitates binding of these compounds to an anion exchange column and

thus allows separation of either the intact GSLs or “desulfo” derivatives after enzymatic

desulfation [78]. Direct analysis of volatile isothiocyanates and nitriles produced from

GSLs by GC/MS can also provide proof of the presence of corresponding GSL in intact

plant. On the other hand, lipophilic bioactive components such as carotenoids are

generally extracted using non-polar solvents such as petroleum ether, hexane, THF,

methanol, ethanol, or combinations of various solvents in different proportions. The

identification is usually carried out by Diode Array Detector (DAD) or MS. Most of the

vitamins are analyzed using standard AOAC methods.

1.4. Different methods for preservation of fresh-cut produce

A wide range of preservation techniques are available in food industry. Disinfectants

presently employed in commercial processing lines, such as chlorine has limited effect

(approx. 1 log reduction) on microbial populations and cannot be relied to eliminate

pathogenic microorganisms like L. monocytogenes. These disinfectants cannot prevent

damage due to elevated respiration and transpiration rates of minimally processed

products. In addition it is believed that they may form carcinogenic derivatives in water

(e.g. chloramines and trihalomethanes) [79]. Therefore, there is a need for research on

42

alternative treatments suitable for use on fresh-cut products. New protocols for

maintaining quality while inhibiting undesired microbial growth for both the production

facilities and distribution chains, are thus needed.

Newer methods available for preservation of RTC products can be classified in two

categories- chemical and physical. Chemical methods involve use of chlorine dioxide,

organic acids, hydrogen peroxide, calcium based solutions, ozone, electrolyzed water and

natural preservatives. Under physical methods of food preservation, high pressure

processing, pulsed white light, ultra violet light, pulsed electric field, oscillating magnetic

fields and ionizing radiation are the recent techniques which are being used widely.

1.4.1. Chemical preservatives

Among the chemical methods, chlorine dioxide with a better oxidation capacity than

chlorine, hydrogen peroxide, a strong oxidizing agent and ozonated water has been used

for reducing microbial populations and shelf life extension of fresh produce. Efficacy of

ClO2 in the inactivation of Listeria monocytogenes and Salmonella typhimurium [80], and

H2O2 solution in reducing microbial populations on fresh-cut bell peppers, cucumber,

zucchini, cantaloupe, and honeydew melon, without alteration in sensory characteristics

[81] have been reported. Although, antimicrobial activity of ozone is widely known, there

is little information available about its efficacy against food borne pathogens. Higher

corrosiveness of ozone, its negative effects on easily oxidizable compounds such as

vitamin C and carotenoids; and initial capital cost for its generation are the main

disadvantages in its use compared to other chemical preservatives.

43

Calcium is extensively used to extend the shelf life of fruits and vegetables. It helps

maintain the vegetable cell wall integrity by interacting with pectin to form calcium

pectate. Different salts of calcium used in food industry include calcium chloride, calcium

lactate and calcium propionate. Antibacterial properties have been reported for calcium

propionate during the treatment of honeydew melon, due to its ability to uncouple

microbial transport processes [82].

Acidic electrolyzed water (pH 2.1-4.5) has a strong bactericidal effect against pathogens

and spoilage microorganisms. It is more effective than chlorine due to its high oxidation-

reduction potential (ORP). A higher effectiveness of electrolyzed water in reducing viable

aerobes than ozone on whole lettuce has been demonstrated [83]. No adverse effects were

noted on the w.r.t. surface color, pH or general appearance of fresh-cut vegetables.

The reluctance of consumers towards the use of chemical preservatives in recent years

has resulted in the use of natural antimicrobials as preservatives. Organic acids such as

lactic, citric, acetic and tartaric acids are used as strong antimicrobial agents against

psychrophilic and mesophilic microorganisms in fresh-cut fruits and vegetables. The

antimicrobial action of organic acids is due to pH reduction in the environment,

disruption of membrane transport and/or permeability, anion accumulation, or a reduction

in internal cellular pH by the dissociation of hydrogen ions from the acid.

1.4.2. Physical methods of preservation

Several physical methods are currently employed for preservation of minimally processed

plant produce. Modified atmosphere packaging (MAP) for example aims at producing

low O2 and high CO2 in the atmosphere surrounding fresh cut produce. These conditions

44

reduce respiration rate thus delaying senescence and extending shelf life. In passive MAP

the package is sealed under normal atmospheric conditions and desired composition is

obtained due to produce respiration and film gas permeability. In active MAP the package

is flushed with a gas mixture of preset composition and once closed, no further control of

the gas composition is exercised. However, expensive equipment and materials (gases

and packaging) are required for MAP generation.

Ultraviolet (UV) light is another physical treatment widely employed in industry. UV

irradiation causes up to 4 log cycle reduction in bacterial, yeast and viral counts by

inducing DNA damage. Major advantage of this technique is the availability of relatively

inexpensive and easy to use equipment. Among the other technologies, treating products

with millisecond pulses (1–20 flashes/sec) of broad spectrum white light, about 20,000

times more intense than sunlight holds promise. Pulsed white light inactivates

microorganisms by combination of photochemical and photothermal effects, requires

very short treatment times and has a high throughput. The above methods, however,

have lower efficiencies due to their lower penetration and are thus mostly used for

surface sterilization.

1.4.2.1. Radiation Processing

Radiation processing is a promising technology for improving the shelf life of fresh-cut

produce. Treatment of food products by ionizing radiation is a physical process

involving direct exposure to electromagnetic γ-rays, X-rays or electron beam for

improvement in food safety and shelf life. It is a non thermal technology that effectively

45

eliminates food-borne pathogens in various foods including fresh vegetables without

compromising the nutritional properties or sensory qualities of food [84].

Gamma irradiation can be employed for inhibition of sprouting, delay in ripening, killing

of insect pests, parasites, pathogenic and spoilage microorganisms (Table 8). Radiation

by its direct effect on macromolecules and indirect effect through radiolysis of water

bring about alteration in the structure of essential bio-molecules present in insects,

parasites, and microorganisms, thus reducing their chances of survival.

In 1980, Joint Expert Committee of Food and Agriculture Organization / International

Atomic Energy Agency / World Health Organization on Food Irradiation concluded “The

irradiation treatment of any food commodity up to an overall average dose of 10 kGy

presents no radiological, microbiological or toxicological hazard.” As a result,

toxicological testing of foods so treated is no longer required. Food irradiation is now

legally accepted in many countries. In 1997, one more expert group constituted by WHO,

reaffirmed the safety of food irradiated at doses above 10 kGy. Consequently, in 2003

Codex Alimentarius Commission revised its Codex General Standard for Irradiated Foods

to set standards for irradiation of foods worldwide. Agreements on Sanitary and

Phytosanitary (SPS) Practices and Technical Barriers to Trade (TBT) under the World

Trade Organization (WTO) have provided for adoption of irradiation as an SPS measure

in international trade under the principle of equivalence. Thus, irradiation can be applied

to overcome quarantine barriers, and to hygienize products for international trade.

The range of dose commonly employed in various food irradiation applications to achieve

different objectives can be classified into three groups: low dose (< 1 kGy), medium dose

46

(1-10 kGy) and high dose (> 10 kGy) applications. Table 9 lists the food items approved

for food irradiation under FSSAI Indian rules.

Table 8. Various applications of food irradiation (Table adapted from FSSAI

(www.fssai.gov.in))

47

Table 9. Food items approved for radiation processing in India under FSSAI rules

1.4.2.2. Advantages of Radiation Processing

• It is an effective alternative to chemical fumigants that endanger human health and

environment.

48

• At recommended doses it maintains fresh-like character, sensory qualities, texture,

nutritive value and appearance of food.

• Does not produce any toxic residues in food.

• Unlike chemical fumigants irradiation can be carried out in pre-packaged foods and

hence no risk of post-irradiation contamination.

• Being highly penetrating and effective, large volumes of foodstuffs can be treated very

efficiently.

• Radiation processing is an eco-friendly treatment and does not pollute environment.

1.4.2.3. Effect of gamma irradiation on shelf-life of RTC vegetables

Shelf life of a fresh produce is determined by its microbial status as well as by its sensory

attributes such as color, texture and flavor (aroma and taste). Gamma irradiation has been

reported to cause minimal modification in these quality attributes of fresh-cut produce.

However, the levels of modification in the quality attributes might vary depending on the

raw material used and radiation dose delivered [85]. Studies conducted on irradiated RTC

vegetables have demonstrated no significant changes in the appearance, color, texture,

taste and overall acceptability after seven days of irradiation (1 kGy) as compared to the

control, when stored at low temperatures (4 - 10 °C) [86]. In contrast, adverse effects of

radiation treatment were observed on color, taste, flavor and texture by the sensory panel,

in pre-cut irradiated tomatoes (lycopersicon syn. L. esculentum), cantaloupe melon

(Cucumis melo), and watermelon (Citrulus lanatus) [87]. 88, Bibi et al. [88] also reported

49

a decreased firmness and lower appearance scores for irradiated tomatoes (L. esculentum)

as compared to controls during storage.

An improvement in the quality of fresh-cut produce by radiation processing has also been

reported by several researchers. Radiation treated products such as conventional chicory

had a better general acceptability when irradiated at doses 1.2 and 2 kGy [89]. The overall

acceptability of carrots was found to be higher up to a radiation dose of 2 kGy as

compared to corresponding controls [90]. The better quality retention in general, has been

attributed to the decreased physiological and microbial decay in the radiation processed

samples. Slight increase in sweetness was observed in radiation-processed carrot (Daucus

carota) at a dose of 2 kGy. Thus, radiation processing may be employed for improving

the shelf life of fresh-cut produce with minimum or no alteration in the sensory quality.

1.4.2.4. Microbial status of irradiated RTC vegetables

One of the main uses of radiation processing is to control the microbial spoilage of food

products. Ionizing radiation causes radiolysis of water producing reactive hydroxyl

radical. Hydroxyl radicals are extremely reactive and attack and damage cellular

components, especially DNA. Damage to genetic material results in inhibition of

microbial growth. Significant reduction in the microbial load consequent to radiation

processing has been widely reported in literature. Irradiation of broccoli and mung bean

sprouts at 1.0 kGy resulted in reduction of approximately 4.88 and 4.57 log CFU/g,

respectively, of a five-strain cocktail of L. monocytogenes. The effects of low-dose

irradiation on the microbiota of pre-cut tomato (lycopersicon syn. L. esculentum) were

investigated by Mohacsi-Farkas et al. [87]. Doses of 1–3 kGy were able to reduce

50

considerably the microbiological contamination in tomato. The low dose irradiation

reduced the viable cell count of Listeria monocytogenes by 2 log-cycles. A dose of 1 kGy

reduced the viable cell number by more than 5 log-cycles. These results indicate that

radiation processing has been successfully employed for microbial decontamination of the

fresh-cut fruits and vegetables. The technology therefore could result in improved shelf-

life of the fresh-cut produce.

1.4.2.5. Effect of radiation processing on phytochemicals

Levels of phytochemicals in plants vary depending on various conditions. Exposure to

radiation sources, wounding, storage at low temperatures, and/or exposure to extreme

temperatures [91] can lead to changes in the nature/content of bioactive phytochemicals.

In terms of radiation exposure, the changes depend on the applied dose (usually low and

medium doses have insignificant effects on phytochemicals), the sensitivity of the

phytochemicals towards irradiation, and the effect of irradiation on precursors that might

be responsible for the production and/or the accumulation of phytochemicals in the plant.

Radiation processing has been shown to either increase or decrease the antioxidant

content of plant produce, depending on the dose delivered, time of exposure and the

nature of raw material to be irradiated.

The enhanced antioxidant capacity of a plant after irradiation is mainly attributed either to

increased enzyme activity (e.g., phenylalanine ammonia-lyase and peroxidase activity) or

to the increased extractability from the tissues [85, 91]. It has been also proposed that

gamma radiation is capable of breaking the chemical bonds of polyphenols, resulting in

51

the release of low molecular weight water soluble phenols, leading to an increase of

antioxidant rich phenolics [92].

A positive correlation between phenolics and antioxidants has been observed in several

cases. The increased antioxidant capacity has been correlated with tissue browning

resulting from an action of polyphenol oxidase (PPO) [93]. Fan et al [94] while working

on lettuce, reported that the free radicals generated during irradiation might act as stress

signals and may trigger stress responses in lettuce, resulting in an increased antioxidant

synthesis. Fan et al. [95] studied the effect of radiation treatment (0, 0.5, 1 and 2 kGy) and

storage (up to 8 d) at 7-8 °C on phenolics content, antioxidant capacity and tissue

browning of romaine lettuce, iceberg lettuce and endive. Their results revealed an

enhancement in the phenolic content and antioxidant capacity of all vegetables at 4 and 8

d after irradiation. The increase in the total antioxidant capacity was attributed to

increased phenolic synthesis.

Reduction in contents of antioxidants has also been reported by various researchers. In

general, the decrease in antioxidants has been attributed to radiation-induced degradation

or the formation of free radicals [96]. Breitfellner et al [97] have reported that gamma-

irradiation (1 to10 kGy) of strawberries lead to the degradation of phenolic acids like

cinnamic, p-coumaric, gallic, and hydroxybenzoic acids.

In recent years there has been an increased demand for RTC vegetables among Indian

consumers. However, very less work has been carried out on the possibility of using

gamma radiation processing to extend the shelf-life of minimally processed Indian

vegetables. It was therefore of interest to determine the feasibility of using radiation

52

processing for extending the shelf life of some commonly used RTC Indian vegetables

and understand changes in major bioactive principles in these products.

1.5. Vegetables selected for the present work

Three popular Indian vegetables namely ash gourd (Benincasa hispida), pumpkin

(Cucurbita pepo) and drumstick (Moringa oleifera) were selected for the present study.

Besides their nutritive value, these vegetables have also been used traditionally for their

pharmacological and medicinal value.

1.5.1. Ash gourd (Benincasa hispida)

Ash gourd (Benincasa hispida), a member of the family Cucurbitaceae and native of

Asia, is one of the most important vegetables grown primarily for its fruits and usually

recognized for its nutritional and medicinal properties. It is also known as winter melon,

ash gourd, ash pumpkin, white gourd, white pumpkin, gourdmelon, tallow gourd, wax

gourd and Chinese watermelon [98]. The average production of the fruit of this green

vegetable is around 18.5 t/ha [99]. India is the second largest producer of this vegetable

after China [100]. The plant is monocious vine type bearing large green fruits that are

either allowed to spread on the ground or trained to climb a support. It takes from 6 to 9

weeks for the yellow flowers to develop after seed germination and the fruit matures after

a period of 2-3 months.

The fruit has been claimed to be a high quality vegetable, based on the Index of

Nutritional Quality (INQ) data [101]. It is a good source for natural sugars, amino acids,

organic acids, minerals and vitamins. Table 10 depicts the proximate composition of

53

immature and mature fruit. Moisture contents are quite high in the fruit accounting for 96

% of the edible portion of the mature fruit. Table 11 summarizes various vitamins and

minerals content reported in mature ash gourd fruit. It contains an appreciable amount of

vitamin C ranging from 1.35 – 68.00 mg/100 g while the content of thiamin is lowest

(0.02 – 0.04 mg/100 g). Potassium (K) and calcium (Ca) are the major minerals present in

ash gourd with concentrations ranging from 77 – 131 mg/100 g and 5 – 23 mg/100 g

respectively. Both these minerals play a crucial role in maintaining the electrolytic

balance of the body fluid.

Table 10. Proximate composition of immature and mature ash gourd fruit (g/100g of

edible portion) (Zaini et al., 2010 [100])

The vegetable finds use as a medicine in the traditional Asian system of medicine system

such as the Ayurveda. It is an important ingredient of “Kusmanda lehyam” (Ayurvedic

medicine) [102]. It a rich source of various functionally important bioactives such as

triterpenes, phenolics, sterols, and glycosides. It has been widely used for the treatment of

epilepsy and other nervous disorders [103]. The juice and extract of the fruit is known to

possess significant anti-ulcer, anti-depressant and diuretic activities, and provide

54

protection against histamine-induced bronchospasm [101]. Common medicinal and

pharmacological properties of the vegetable are listed in table 12.

Table 11. Vitamins and minerals profile of mature ash gourd fruit (100 Zaini et al., 2010)

Owing to its popularity, many studies on ash gourd have been reported. Some volatile

compounds have been identified in the ash gourd with aliphatic alcohols and carbonyl

compounds as the major class [104]. Volatile compounds of the beverages prepared from

this vegetable were analyzed by Sikorski [105] and Wu et al. [106]. Pyrazines were found

to be major compounds detected in beverages which were presumably formed by the

Maillard reaction during extraction of juices.

55

Table 12. Some common medicinal and pharmacological properties of different parts of

ash gourd (100 adapted from Zaini et al., 2010 [100])

The vegetable is sold in the whole as well as in the sliced form. The intact vegetable can

be stored for several days. However, the large size of the vegetable creates problem for its

storage in supermarket and retail outlets, thus requiring some degree of preparation. Once

cut, its quality deteriorates within a day or two. The high moisture content and presence

of macronutrients such as sugars make it vulnerable to spoilage by microorganisms

limiting its marketability. Recently, researchers have examined shelf life extension of cut

56

ash gourd using a combination of chemical pretreatment and partial dehydration [107].

However, after the treatment, the product was not fresh. It was therefore of interest to

determine the feasibility of using gamma irradiation for improving the shelf life of ready-

to-cook ash gourd and assessing the effect of such a processing technique on its various

physico-chemical properties.

1.5.2. Drumstick (Moringa oleifera)

Moringa oleifera is the most widely cultivated species of a monogeneric family, the

Moringaceae, and is native to the sub-Himalayan tracts of India, Pakistan, Bangladesh

and Afghanistan. It is also called as the horseradish, drumstick, benzolive, kelor,

marango, mlonge, moonga, mulangay, saijan, sajna or Ben oil tree. It is a slender

softwood tree that branches freely, and can be extremely fast growing. Although it can

reach heights in excess of 10 m (33 ft), it is generally considered a small- to medium-size

tree. The fruits are tri-lobed capsules, and are frequently referred to as “pods.”

Commercial production of immature pods for processing is a large industry in India with

about 1.2 million MT (metric tons) produced annually on 38,000 ha.

It is an important food commodity which has received enormous attention as the ‘natural

nutrition of the tropics’. Moringa trees have been used to combat malnutrition, especially

among infants and nursing mothers. Almost every part of the plant - the leaves, fruit,

flowers and pods are used as a highly nutritive vegetable in many countries including

India, Pakistan, Philippines, Hawaii and many parts of Africa [108]. The proximate

composition of drumstick pods is shown in Table 13. The high concentrations of ascorbic

57

acid, oestrogenic compounds and β-sitosterol, iron, calcium, phosphorus, copper,

vitamins A, B and C, α-tocopherol, riboflavin, nicotinic acid, folic acid, pyridoxine, β-

carotene, protein, and in particular essential amino acids such as methionine, cystine,

tryptophan and lysine present in drumstick leaves and pods make it a virtually ideal

dietary supplement [109].

Apart from nutritional values, it has numerous medicinal uses, which have long been

recognized in the Ayurvedic and Unani systems of medicine [110]. The various medicinal

and pharmacological activities of different parts of the plant are summarized in Table 14.

Almost all parts of this plant : root, stem, bark, gum, leaf, fruit (pods), flowers, seed and

seed oil have been used for various ailments in the indigenous medicine of South Asia,

including the treatment of inflammation and infectious diseases along with

cardiovascular, gastrointestinal, hematological and hepatorenal disorders [111, 112].The

medicinal properties of the plant have been attributed to the presence of bioactive

phytochemicals including phenolic compounds, and a unique group of compounds called

glucosinolates and isothiocyanates [113, 58]. They have been reported to possess

hypotensive, anticancer, and antibacterial activity. The compounds responsible for these

properties include 4-(4'-O-acetyl-a-L-rhamnopyranosyloxy)benzyl isothiocyanate [1], 4-

(α-L-rhamnopyranosyloxy)benzyl isothiocyanate [2], niazimicin [3], pterygospermin [4],

benzyl isothiocyanate [5], and 4-(α-L-rhamnopyranosyloxy)benzyl glucosinolate [6]

(Figure 8). While these compounds are relatively unique to the Moringa family, it is also

rich in a number of vitamins and minerals as well as other more commonly recognized

phytochemicals such as the carotenoids (including β-carotene or pro-vitamin A).

58

Table 13. Proximate composition and minerals and vitamins (content per 100 g of edible

portion) in drumstick pods

59

Table 14. Medicinal and pharmacological activities of different parts of drumstick tree

It has been estimated that about one-fourth of all Moringa oleifera produce harvested is

spoiled before consumption. Therefore, there are increasing interests to preserve the

vegetable due to its medicinal and therapeutic properties [114]. Studies have been

attempted to preserve the shelf-life of leaves and pods of this vegetable using various

preservation techniques [115]. Treatments with germicide, salt and turmeric were

employed in various combinations and it was followed with dehydration by sun drying,

oven drying or wind drying. Microbiological and physico-chemical analyses were

performed on the samples for a storage period of 4 months. The employed preservation

60

techniques involving dehydration were found to effectively extend the shelf life of

Moringa oleifera without alteration in the nutritional value. However, no reports exist on

the preservation or shelf-life extension of fresh-cut drumstick pods. It was therefore of

interest to study the effect of gamma irradiation on the shelf-life and quality attributes of

the fresh-cut drumstick.

Figure 8. Glucosinolates reported in drumstick (Moringa Oleifera). 4-(4'-O-acetyl-a-L-

rhamnopyranosyloxy)benzyl isothiocyanate [1], 4-(α-L-rhamnopyranosyloxy)benzyl

isothiocyanate [2], niazimicin [3], pterygospermin [4], benzyl isothiocyanate [5], and 4-

(α-L-rhamnopyranosyloxy)benzyl glucosinolate [6]

61

1.5.3. Pumpkin (Cucurbita pepo)

Pumpkin (Cucurbita pepo) belongs to the Cucurbitaceae family which includes melons,

squashes, cucumbers and gourds. It is defined botanically as a fruit although commonly

regarded as a vegetable by the consumer. The pumpkin varies greatly in shape, ranging

from oblate to oblong, with smooth, slightly ribbed skin and deep yellow to orange

coloration. It is widely grown and consumed in many countries around the world [116]

including India. The biggest international producers of pumpkins include the United

States, Canada, Mexico, India, and China. According to estimations by the Food and

Agriculture Organization of the United Nations (FAO), world production of pumpkins in

2007 was over 20 million tons, especially in China, India, Russia, United States, and

Egypt [117].

In recent years, pumpkin has received considerable attention because of the nutritional

and health protective value. The vegetable significantly contributes to nutrition as it is a

rich source of minerals. It also contains a high content of carotenoids in its flesh,

including β-carotene, lutein, and violaxantine. It also possesses several phytoconstituents

belonging to the class of alkaloids, flavonoids, and fatty acids such as palmitic, oleic and

linoleic acids [118]. In addition, many cultivars of pumpkin are characterized by a high

content of vitamin C [118]. It possesses low calorific value (15–25 kcal in 100 g) and due

to the presence of numerous, easily digestible nutrients it has become an important

component of slimming diets. It regulates metabolism, lowers glucose level in blood, and

possesses detoxicating as well as dehydrating properties. Other uses attributed to pumpkin

species include defense against cancer [119] and diabetes and for internal as well as

62

external management of worms and parasites. Tables 15 and 16 represent the proximate

composition and medicinal properties of the pumpkin.

Due to the large size of the vegetable it is difficult to store thus restricting marketability.

Although, the intact vegetable can be stored for several days; once cut, it is highly

perishable and deteriorates within 1-2 days. The high moisture content and presence of

macronutrients such as sugars make it vulnerable to spoilage by microorganisms limiting

its marketability. Recently, the shelf-life of cut pumpkin under modified atmospheric

packaging (30% CO2/ 70% N2; 100% CO2 and vacuum) and stored at refrigeration

temperature was examined [120]. 100% CO2 was inferred to be the best by these workers

for a storage period of 5 days, with total microbial counts of 7.7 x 103 CFU g-1 and yeast

and mould counts of approx 10 CFU g-1 after 5 days. However, to the best of our

knowledge, efficacy of radiation processing in enhancing the shelf-life and quality of

fresh-cut pumpkin has not been investigated so far.

63

Table 15. Proximate and minerals and vitamins content in pumpkin pulp (per 100 g of

edible portion)

(Table adapted from USDA Nutrient Database )

64

Table 16. Medicinal and pharmacological activities of pumpkin

65

1.6. Scope of the work - Aims and objectives

Importance of convenience foods has increased considerably in recent times. These

include fresh-cut ready-to-eat (RTE) fruits and ready-to-cook (RTC) vegetables.

However, they have very limited marketing due to short shelf life. Gamma irradiation has

been demonstrated as a promising tool for improving the shelf-life of such fresh-cut

produce. Radiation processing is reported to act as an abiotic stress for the living tissues

such as fresh-cut fruits and vegetables, thereby resulting in changes in the post harvest

physiology of the product. Consumers and food safety advocates are therefore worried

about the nutritional and chemical quality of such radiation treated produce.

One of the main mechanisms by which plants protect themselves against adverse

environmental abiotic and biotic stress is production of reactive oxygen species (ROS).

However, emerging data show that ROS production in certain situation can also

contribute to the improved physiology and increased fitness of plants. The effect of

different postharvest abiotic stresses (i.e., wounding, UV-light, hyperoxia, and the

exogenous application of certain compounds or hormones) on the accumulation of

phenolic compounds in fruits and vegetables has been evaluated in several studies.

Nevertheless, little is known on the physiological basis for the accumulation of

phytochemicals/antioxidants as a result of radiation processing in vegetables of Indian

origin. Increasing the scientific knowledge in this area is critical to envisage strategies

that permit the effective use of crops as bio-factories of nutraceuticals.

Vegetables during processing such as cutting and cooking give rise to characteristic

aroma to the cuisine. Presence of glycosidic precursors of volatile aroma compounds has

66

been well established in the plant kingdom. These precursors are known to undergo

hydrolytic or enzymatic breakdown during processing, resulting in the release of volatile

aroma compounds, and hence enhanced aroma quality. The same hypothesis may be

extended to explain the released aroma during processing in vegetables, however, it needs

to be further explored. Few reports exist on the nature of aroma compounds present in

most of the vegetables, especially of Indian origin. Radiation processing is also reported

to result in breakdown of aroma glycoconjugates resulting in liberation of free aroma and

thus an enhancement in overall flavor quality. No studies exist on the effect of irradiation

on flavor precursors in majority of the vegetables. As aroma quality plays a critical role in

deciding the consumer acceptance of a food product. The effect of radiation processing on

the aroma constituents of fresh-cut produce thus needs to be investigated.

There is an increasing interest in the bioactive components of native plants and

identifying their role as functional foods. This has resulted in a shift towards identifying

native plant foods possessing novel organic molecules with unique bioactivities. In this

context use of Indian vegetables as medicinal ingredients in Ayurveda has been in

practice since early times. Despite being a treasure house of active constituents, very few

studies exist on the nature of the constituents present in majority of the Indian vegetables.

Although, several reports exist in the literature regarding chemical composition, aroma

and medicinal attributes of the above mentioned vegetables, extensive work is required to

study the effect of gamma irradiation on various phyto-chemical aspects of these

vegetables in order to understand the sensory and nutritional status of the radiation

processed product.

67

Therefore, the major objectives of the thesis are as follows:

To study the effect of gamma irradiation on the shelf life of ready-to-cook (RTC)

products of selected Indian vegetables namely ash gourd (Benincasa hispida),

pumpkin (Cucurbita pepo) and drumstick (Moringa oleifera).

Physico-chemical characterization of the developed shelf stable radiation processed

RTC products.

Isolation and identification of aroma compounds, key odorants in the above mentioned

vegetables and to study the effect of radiation processing and storage on the aroma

profiles.

To study the effect of radiation processing on some bioactive principles from the

selected vegetables.

68

CHAPTER 2

MATERIALS AND METHODS

69

2. 1. Materials

2.1.1 Plant material

Vegetables - ash gourd (Benincasa hispida), bottle gourd (Lagenaria siceraria),

pumpkin (Cucurbita pepo), lady finger (Abelmoschus esculentus) and drumstick

(Moringa oleifera) were procured from a local grower around Mumbai, India. The

vegetables were brought to the laboratory within 12 h after harvesting and immediately

stored at refrigerated temperatures (10 ± 1 °C) before processing.

2.1.2. Chemicals and materials

Amberlite XAD-2 resin was procured from Sigma-Aldrich, USA. Reverse phase (C18)

solid phase extraction cartridges with 6 mL volume and 0.5 g active phase were

procured from Supelco, USA. Solid phase micro extraction (SPME) fibres made up of

Poly dimethoxy siloxane-divinylbenzene-carboxen (PDMS/DVB/CAR) with film

thickness of 0.65 μM and 1 cm length, were bought from Supleco, USA.

Methanol and dichloromethane was procured from Merck India Pvt. Ltd. Diethyl ether,

n-hexane and n-butanol was purchased from S.D. Fine Chemicals, India. All solvents

used were of analytical grade and were redistilled before use. HPLC grade solvents such

as acetonitrile, methanol, glacial acetic acid and o-phosphoric acid were procured from

Merck Ltd., Germany.

PCA, PDA, Murashige & Skoog Medium, (With Vitamins; without CaCl2, Sucrose,

IAA, Kinetin and Agar) and DPPH was purchased from HiMedia Laboratories, India.

Folins-ciocalteu reagent was purchased from Merck, India. Boric acid and disodium

hydrogen-o-phosphate were from Qualigens Fine Chemicals and sodium dihydrogen-o-

70

phosphate was procured from Thomas Baker Ltd. Potassium ferricyanide (K3[Fe(CN)6])

and sodium bicarbonate (NaHCO3) were from BDH Laboratory Chemicals and Chemco

Fine Chemicals, respectively. Hydrogen peroxide and m-phosphoric acid were procured

from S.D. Fine Chemicals Ltd.

Various standard compounds used i.e. acetoin, acetobromo-D-glucose, phenolic acids,

carotenoids, enzyme preparations – pectinase (≥ 5 U/mg protein), myrosinase (≥ 100

U/g) and sulfatase (≥ 10000 U/g) were bought from Sigma Aldrich, USA.

2.2. Methodology

2.2.1. Development of ready-to-cook (RTC) vegetables using radiation processing

2.2.1.1. Preparation of RTC vegetables

Whole ash gourd, bottle gourd, pumpkin, lady finger and drumsticks were washed in

running tap water to remove adhered dust. After washing, ash gourd, bottle gourd and

pumpkin were hand peeled and cut into small pieces of dimensions 2.5 cm × 2.5 cm ×

0.7 cm with a sharp sterile knife. The lady fingers and drumstick pods were transversely

cut in to pieces of length 1.5 and 4.0 cm, respectively. The cut pieces of each vegetable

were separately randomized before packaging, and were packed (100 g) into polystyrene

trays (inner dimensions: 9 cm × 9 cm × 2.5 cm). The trays were then over-wrapped all

around with cling film (Flexo film wraps ltd., Aurangabad, Maharashtra, India) and

sealed to avoid any leakage. Film used in the present study had thickness of 8–10 µm

and permeability to oxygen and carbon dioxide as 1.7 and 10.3 cm3m

−2 s

−1 Pa

−1

respectively (data as provided by supplier).

71

2.2.1.2. Radiation processing and storage

Packaged cut vegetables were subjected to various radiation doses (0.5, 1.0, 1.5, 2.0 and

2.5 kGy) in a cobalt-60 irradiator (GC-5000, BRIT, Mumbai, India) having a dose rate

of 1.64 Gy/s. Irradiator was calibrated by Fricke dosimeter before start of experiment

and had a dose uniformity ratio of 1.2. Irradiated samples were stored in the dark at 10 ±

1 °C. In preliminary studies on RTC products carried out in our laboratory, the optimal

storage temperature was found to be 10 °C. Storage at a lower temperature (4 °C)

induced chilling injury resulting in low aroma scores whereas at a higher temperature of

15 °C, physiological as well as microbial spoilage was very rapid resulting in shorter

shelf life. Non-irradiated samples of respective vegetables stored at 10 ± 1 °C were used

as controls during the entire storage period. Three replicates were prepared for each

dose and storage day. The samples were examined at different time intervals during

storage. During initial investigations, it was observed that cut bottle gourd developed

browning immediately after cutting. In radiation processed cut lady finger, the sticky

mucilaginous secretions increased with radiation dose, thus making irradiated lady

finger unacceptable by the sensory panel. The treatment of anti-browning reagents in

case of bottle gourd and some chemical treatments in case of lady finger prior to

irradiation could help in their shelf-life improvement. It was therefore inferred that

radiation processed bottle gourd and lady finger were not amenable for development of

RTC products and hence they were not studied further.

72

2.2.1.3. Optimization of quality parameters

Experimental design and subsequent analysis of results were performed by a software

Design Expert 8.0 (State-ease Inc., U.S.A.). Control and radiation-treated samples were

analysed on day 0, 5, 8, 12 and 15 after packaging for ash gourd and drumstick, whereas

for pumpkin, the analyses were conducted on days 0, 7, 14, 21 and 28 after packaging.

A full factorial experimental design was employed with gamma radiation dose and

storage time as variable factors to be optimized [121]. Experiments were planned at

different radiation doses (0, 0.5, 1.0, 1.5, 2.0 and 2.5 kGy). Three replicates were

prepared for each dose and storage day. Parameters studied for analyzing product

quality were microbial load, color, texture and sensory acceptability.

Models for individual responses were generated by fitting experimental data into third

order polynomial equations and then removing insignificant terms by backward

regression method as reported earlier [122]. In this approach, model terms with highest

values of partial probability (p-value) are removed first and the process is stopped when

the p-value of next term out satisfies the specified alpha out criterion. Value of alpha out

was kept 0.1 which leads to an overall model with terms significant at 0.05 levels. The

models were analyzed by analysis of variance (ANOVA) and the coefficient of

determination (R2) was used to quantify the predictive capability of the model. Fitted

polynomial equations were then plotted in order to visualize the interrelationship of

response and experimental levels of each factor. Optimal conditions were then derived

using numerical optimization process. Criteria for desired microbial and sensory

73

qualities were provided and model equations solved to obtain optimum processing

conditions.

2.2.1.3.1. Microbial quality

Standard methods were used to enumerate total microbial load present in the RTC

vegetables at each sampling time and treatment for the intended duration of storage [123].

Enumeration of total aerobic mesophilic bacteria was carried out by pour-plate method on

plate count agar (Himedia, Mumbai, India). Incubation was done at 37 °C for 24 h. Total

yeast and mould count was performed by pour-plate method on potato dextrose agar

(Himedia, Mumbai, India) supplemented with 0.01 g L-1

tartaric acid to lower pH of the

medium to 3.5. Plates were incubated at 37 °C for 48 h. Microbial counts were expressed

as log10 CFU g-1

. Each analysis was performed in triplicate.

2.2.1.3.2. Sensory quality

Cut pieces of each vegetable were cooked separately in boiling water for 5 min and

presented to the assessors in white trays for sensory assessment in different sessions.

Hedonic testing was carried out for each vegetable by 15 panelists of trained sensory

panel using a 9-point scale with 1, dislike extremely or not characteristic of the product

and 9, like extremely or very characteristic of the product [123]. Parameters evaluated

were colour, aroma, texture, taste, aftertaste and overall acceptability. Irradiated and

stored samples were compared with fresh-cut control samples of the respective vegetables

on each day of study to analyze all the parameters.

74

Color and firmness were also assessed instrumentally for all the samples prepared.

Surface colour values were evaluated using a colorimeter (CM- 3600d Konica Minolta

sensing Inc., Japan) by measuring the three Commission Internationale de l’Eclairage

(CIE) coordinates, L (lightness), a (−green, +red), and b (−blue, +yellow). The instrument

was calibrated using a white tile supplied along with the equipment. Six pieces of each

vegetable were selected randomly from every packaged tray for color measurement and

results represent their average. Firmness was measured using a texture analyzer

(TA.XTPLUS, Stable Microsystems Ltd., Surrey, England) with a 980 N load cell,

equipped with a cone probe (P/45C s/s batch No. 3889) for ash gourd and pumpkin, while

a needle probe (P/2N s/s batch No. 10451) was employed for piercing test through the

drumstick samples. The test was designed with a trigger force of 0.147 N and test speed

of 0.5 mm/s. The peak force required to penetrate samples was referred as a measure of

firmness of the sample. Data were collected for six replicates from each tray and results

were expressed as their average.

At optimized processing conditions, sensory evaluation was also performed by

quantitative descriptive analysis (QDA) [124] along with hedonic analysis. A trained

panel consisting of 10 members, 6 males and 4 females assessed the samples using

unstructured 150-mm scale. The sensory attributes for each of the vegetable studied were

finalized during prior training sessions conducted. The sensory attributes assessed for

pumpkin were color (yellow), aroma (pumpkin-like, irradiated and musty), and taste

(pumpkin like and irradiated); for ash gourd the attributes were color (green), texture,

aroma (ash gourd-like, buttery irradiated and musty), and taste (ash gourd like and

75

irradiated); and that for drumstick were color (green, brown), texture, aroma (drumstick-

like, green, irradiated and musty), and taste (sweet, drumstick like and irradiated).

Assessments were repeated twice and sensory data were collected by measuring distance

(mm) from the origin. Non-irradiated ash gourd samples exhibited visible browning after

a storage period of 5 d, while browning was inhibited in the radiation processed samples

throughout the intended storage.

2.2.2. Characterization of the developed products at optimized parameters

2.2.2.1. Nutritional quality

2.2.2.1.1. Preparation of methanolic extract

Each vegetable (10 g) was separately homogenized and extracted twice with 20 mL

distilled methanol in an omnimixer (Sorvall, U.S.A). The extract was centrifuged at 3000

x g for 30 min and the supernatant thus collected was made to a final volume of 50 mL by

adding methanol. Aliquots were used for determining antioxidant status in terms of total

phenolics and DPPH radical scavenging potential.

2.2.2.1.2. Radical scavenging activity

DPPH radical scavenging assay was used to evaluate total antioxidant activity of the RTC

vegetables according to the procedure of Wen et al. [125]. The final reaction mixture used

for the study was 3 mL (25 µL methanolic extract + 1 mLof DPPH solution (200 µM in

methanol) + 1975 µL methanol). After incubation under dark conditions for 30 min,

absorbance was measured at 516 nm. Total antioxidant activity was expressed as the

76

Gallic acid equivalent mass per mass of the vegetable studied (mg kg−1

). [GAE (mg kg−1

)

= (((% Inhibition – 3.866)/15.67) X 10 X 40)/10); R2 = 0.97]

2.2.2.1.3. Total phenolic content

Total phenolic content was evaluated in accordance with the Folin–Ciocalteu procedure

[126]. Part of the methanolic extract obtained as above (Section 2.2.8.1) was treated with

polyvinyl polypyrrolidone (10 gL-1

, PVPP, Sigma–Aldrich, USA) to remove phenolic

compounds from extract. PVPP was purified as per the procedure detailed earlier [127].

The mixture was then incubated overnight in an orbital shaker at 25 °C at 2.5 oscillations

per s. PVPP was then removed by centrifugation at 21,000 × g for 10 min at 4 °C. The

supernatant was collected and the sediment (PVPP–polyphenol complex) was discarded.

The absorption of the supernatant and original extract was measured at 725 nm using

UV–visible spectrometer (Helios α, Thermofisher Scientific, USA) in accordance with

the Folin–Ciocalteu procedure. The content of total phenolics in the vegetable was

determined by the difference between phenolic content obtained before and after PVPP

treatment and then expressed as the Gallic acid equivalent mass per mass of the fresh

vegetable as mg kg−1

. [GAE (mg kg−1

) = ((O.D + 0.017)/0.048); R2 = 0.99]

2.2.2.1.4. Ascorbic acid content

Total vitamin C content of the processed vegetables was estimated in accordance with

standard AOAC official microfluorometric method [128]. Cut vegetables (20 g) were

extracted separately with 20 mL of freshly prepared extracting solution containing

metaphosphoric acid (0.3 mol L−1

) and acetic acid (1.4 mol L−1

). Homogenate was then

centrifuged at 16,000 × g for 15 min at 4 °C and the supernatant was treated with

77

activated charcoal (20 g L−1

) with vigorous shaking to convert ascorbic acid into

dehydroascorbic acid. The mixture was again centrifuged at 21,000 × g for 10 min at 4 °C

to remove charcoal. Aliquots of this supernatant (500 µL) were added to a solution

containing equal volumes of boric acid and sodium acetate (3% boric acid in 3.67 mol L−1

sodium acetate solution). The solution was allowed to stand for 15 min and the total

volume was then adjusted to 10 mL using milli Q water. This was designated as blank

solution. To prepare the sample solution, another aliquot of 500 µL was mixed with an

equal volume of sodium acetate solution (3.67 mol L−1

) and the total volume was adjusted

to 10 mL using milli Q water. A 0.4 mL aliquot of both (sample and blank) solutions was

separately treated with 1 mL of o-phenylenediamine solution (0.02 %) (Sigma Chemical

Co. Ltd., St. Louis, USA), vortexed and incubated for 35 min at ambient temperature (25

± 2 °C). Ascorbic acid reacts with o-phenylenediamine to form a fluorescent conjugate.

Total conjugate formed was measured (EXλ 350 nm; EMλ 430 nm, Bandwidth 5 nm)

using CS-5000 fluorimeter (Shimadzu Corporation, Japan). The same process was

followed for standard ascorbic acid solutions of known concentration (0.1–0.0015 %) to

obtain a standard curve. Linear regression was then used to determine the concentration

of ascorbic acid in each sample. The content of vitamin C per mass of ash gourd was

expressed in mg kg−1

. All analyses were carried with three independent samples each

analyzed in triplicate.

2.2.2.2. Aroma quality

2.2.2.2.1. Isolation and identification of free aroma compounds

2.2.2.2.1.1. Isolation of free aroma

78

Optimization of procedures for isolation of free aroma was carried out for the selected

vegetables (ash gourd, pumpkin and drumstick). Free aroma volatiles were isolated using

three different techniques – simultaneous distillation extraction (SDE), high vacuum

distillation (HVD) and solid phase microextraction (SPME). Extraction using SDE and

HVD was essentially performed as per procedure described earlier [129, 130] while

procedure followed for SPME extraction was as per Sagratini et al. [131]. A flow diagram

depicting different methodologies followed for extraction of free aroma volatiles is

demonstrated in Figure 9.

Figure 9. A schematic representation of the different methodologies adopted for the

isolation and analysis of free aroma volatiles.

79

A brief decription of the main methodologies adopted for aroma isolation is provided

below.

A) Simultaneous Distillation Extraction (SDE): Ash gourd, pumpkin and drumstick,

200 g each was cut into small pieces and separately subjected to SDE [132] for 2 h using

peroxide free diethyl ether (AR Grade, S.D. Fine-Chem. Ltd., Mumbai, India) as

extracting solvent. The solvent was then removed by passing a slow stream of nitrogen

to obtain the volatile oil.

B) Solid Phase Microextraction (SPME): To each cut vegetable sample (50 g) was

added 10g NaCl and 10 mL distilled water and then homogenized (B400, Buchi,

Switzerland) while maintaining chilled conditions. The homogenate obtained was

filtered through double layered washed & dried muslin cloth. The extract obtained was

centrifuged at 12,000 rpm at 20 °C for 15 min. To the sample, 2-octanol (1.25 μg) was

further added as internal standard. Samples were equilibrated for 30 min at 30 °C with

continuous stirring and headspace was subsequently extracted by preconditioned

PDMS/DVB/CAR fibres (Supleco, USA) for 20 min at 30 °C. After extraction, fibres

were desorbed (270 °C) in the injection port of the GC/MS equipment. Quantification

was performed by comparing peak areas of compounds with that of internal standard

and results obtained as μg kg-1

.

C) High vacuum distillation (HVD): High vacuum distillation was carried out

according to the procedure reported in literature [129, 130]. Cut ash gourd (200 g) was

frozen in liquid nitrogen before use. The sample was placed in a glass tube (5 cm i.d., 25

cm length) and then connected to a distillation unit maintained under vacuum (1 x 10-3

80

torr). The distillate collected in a receiving tube (3 cm i.d., 20 cm length) was

maintained at low temperature using liquid nitrogen. The isolate (50 ml) was extracted

with diethyl ether (3 x 5 ml). Solvent was removed by a slow stream of nitrogen to

obtain oil that was subjected to GC/MS analysis.

2.2.2.2.1.2. GC–MS analysis of free aroma compounds

The aroma concentrates obtained from SDE, HVD and SPME were subjected to GC–

MS analysis on a Shimadzu GC–MS instrument (Shimadzu Corporation, Kyoto, Japan)

equipped with a GC-17A gas chromatograph and provided with a DB-5 (J&W

Scientific, California, USA) capillary column ((5 %-phenyl)-methylpolysiloxane,

length, 30 m; i.d., 0.25 mm and film thickness, 0.25 µm). The operating conditions

were: column temperature programmed from 60 to 200 °C at the rate of 4 °C/min, held

at initial temperature and at 200 °C for 5 min. and further to 280 °C at the rate of 10

°C/min, held at final temperature for 20 min; Injector and interface temperatures, 210

and 280 °C, respectively; carrier gas helium (flow rate, 0.9 ml/min); ionization voltage,

70 eV; electron multiplier voltage, 1 kV. Peaks were tentatively identified by comparing

their mass fragmentation pattern with that of standard compounds wherever available,

from standard spectra available in the spectral library (Wiley/NIST Libraries) of the

instrument as well as by comparing RI (retention index) values of the compounds with

literature data. The content of the identified compounds in the essential oil obtained

from SDE and HVD methods were estimated from a standard curve (R2 = 0.99) of

concentration versus peak area prepared using different concentrations of standard

81

acetoin (0.01–20 µg/µl) for all the three vegetables studied. The contents of various

aroma compounds identified were expressed as µg/kg of the fresh weight of the

vegetable.

2.2.2.2.2. Isolation and identification of bound aroma precursors

Optimization of procedures for isolation of bound aroma precursors was carried out for

all the three selected vegetables. Aroma glycosides were isolated using XAD column

and solid phase extraction (SPE) using C18 cartridges. Extraction using XAD column

was essentially performed as per procedure described earlier by Arul et al. [133] while

procedure followed for SPE extraction was as per Solis et al. [134]. A flow chart

depicting different methodologies followed for extraction of bound flavour precursors is

demonstrated in Figure 10.

The methodologies adopted for extraction of bound aroma glycosides are briefly

described below:

2.2.2.2.2.1. Preparation of extracts

For isolation of bound aroma glycosides, vegetable extracts were prepared using two

different approaches as described below:

A) Cut vegetables (50 g) were homogenized with 200 mL of methanol using a high

speed mixer (Omnimixer, Sorvall, USA) for three min at a speed corresponding to the

position of the knob at position four. The resultant slurry was filtered through a Buchner

funnel under suction. Residue obtained was then further extracted twice using same

solvent. Extracts from all three extractions were pooled and evaporated to dryness under

vacuum (40 mbar, 40 °C) using a rotary evaporator (Buchi, Switzerland). Dried residue

82

was then dissolved in 150 mL of distilled water. This was designated as aqueous

methanol extract.

B) In second approach, 150 g of cut vegetables were added to 50 ml of distilled water

and then homogenized using a high speed homogenizer (B400, Buchi, Switzerland).

Resulting slurry was then centrifuged (5810R, Eppendorf, Germany) at 12000 rpm for

15 min. Clear juice thus obtained was directly used for further extraction of aroma

glycosides.

83

Figure 10. A schematic representation of the different procedures followed for isolation

and analysis of bound aroma precursors.

2.2.2.2.2.2. Isolation of aroma glycosides

Bound aroma glycosides from the vegetables were extracted using two different

extraction methods which are briefly described below:

A) XAD: Aqueous methanol extract of each of the vegetable was subjected separately

to XAD column for isolation of aroma glycosides. XAD resin was packed in glass

column of 2.5 cm I.D. up to a length of 18 cm using methanol. Column was then washed

using 200 mL of methanol followed by 200 mL of diethyl ether. The column was finally

equilibrated with 200 mL of distilled water. Vegetable extracts (200 mL) were

separately loaded on XAD columns with a flow rate of 1.5 mL min-1

. Post loading,

column was washed with 200 mL deionized water and diethyl ether to remove polar and

mid-polar impurities. Subsequently, elution with 200 mL methanol yielded the bound

aroma precursors or aroma glycosides. Methanol fraction was further evaporated to

dryness using rotary evaporator and the residue obtained was finally dissolved in 10 mL

of distilled water for further analysis.

B) Solid Phase Extraction (SPE): SPE cartridges were washed with 10 mL methanol

and then further equilibrated by eluting with 10 mL of distilled water. Vegetable juice

(15 mL) was then loaded on the cartridge while maintaining a flow rate of 1 mL min-1

.

Cartridge was subsequently, washed again with 10 mL of distilled water followed by 10

84

mL of diethyl ether and the bound flavor precursors were then eluted with 10 mL of

methanol.

2.2.2.2.2.3. Identification and quantification of bound aroma

The extracts obtained using XAD and SPE extraction were either acid hydrolyzed using

1 N HCl or subjected to enzymatic hydrolysis using pectinase (Figure 10). Detailed

procedure followed for hydrolysis is as follows:

A) Acid hydrolysis: Extract containing aroma glycosides (10 mL) obtained from XAD

as described in section 2.2.2.2.2.2 was made to 1 N with concentrated HCl. This

solution was then heated at 80 °C for 1 h in a stoppered conical flask. The hydrolysate

was extracted with diethyl ether (3 x 20 ml) and the organic layer containing free

aglycones was repeatedly washed with distilled water till free of acid. The organic layer

was dried over sodium sulphate and then concentrated to less than 1 mL volume using

Kuderna-Danish concentrator (Supelco, USA). Extract was finally concentrated to a

volume of 100 μL using a gentle stream of nitrogen and then injected (1 μL) into

GC/MS equipment for identification of free forms.

B) Pectinase hydrolysis: Methanol fraction obtained from SPE containing aroma

glycosides obtained as described in section 2.2.2.2.2.2 was concentrated to dryness

using rotary evaporator and the residue was dissolved in 15 mL of citrate phosphate

buffer (0.1 M, pH 5). Pectinase preparation (500 μL) was added into this solution and

the samples were kept for incubation at 37 °C for 48 h. Sample hydrolyzed with

pectinase was taken into a 40 mL SPME vial, 4.5 g of NaCl added and then equilibrated

at 30 °C for 45 min. The free aroma compounds were extracted with a preconditioned

85

(270 °C, 10 min) SPME fibre (PDMS/DVB/CAR). Extraction was carried out by

exposing SPME fiber in sample headspace for 20 min at 30 °C. Post extraction, fiber

was desorbed in split/splitless port of GC/MS kept at 270 °C and the analysis was

carried out in splitless mode. Before SPME extraction, 2-octanol (1.25 μg) was added as

internal standard. Each analysis was carried out in triplicate for quantification.

Samples were analyzed on a GC/MS (QP5050A, Shimadzu, Japan) instrument equipped

with RTX-5 column (5% diphenyl-dimethyl-polysiloxane, 0.25 μm I.D., 30 m length,

Restek corporation, USA). The conditions for analysis were similar as described in

section 2.10.1.2. Data was acquired in scan mode from m/z 40 to 350. Peaks were

identified by comparing their mass fragmentation pattern and Kovat’s indices with that

of standard compounds as well as from the data available in the spectral (Wiley/NIST)

libraries of the instrument. Quantification was performed by comparing peak areas of

compounds with that of internal standard and results obtained as μg kg-1

.

2.2.2.2.3. Chemometric analysis of the aroma data

Data obtained for free as well as bound aroma (TIC v/s retention time) was exported in

ascii format to Microsoft Excel using the inbuilt software of GC/MS. The

corresponding ascii data were then separately subjected to PCA for analyzing changes

as a result of radiation treatment and storage. Score plots were drawn and results were

visually analyzed. Data obtained for principal components was further analyzed using

ANOVA and means comparison was performed using Duncan’s multiple range test. All

the statistical analysis was carried out using XLSTAT 2012 software (Addinsoft Inc.

U.S.A.).

86

2.2.2.2.4. Identification of aroma impact compounds

2.2.2.2.4.1. GC-O analysis

The aroma concentrates of the three vegetables obtained from SDE and in SPME mode

were subjected to GC-O analysis on a Shimadzu GC-MS instrument (Shimadzu

Corporation, Kyoto, Japan) equipped with a GC-17A gas chromatograph, provided with

a DB-5 (J&W Scientific, California, USA) ((5%-Phenyl)-methylpolysiloxane, length, 30

m; id., 0.25 mm and film thickness, 0.25 mm) and an olfactory detection port (ODP-2,

Gerstel, Germany). A fixed splitter (1:1) was used at the end of capillary column. One

part of the column flow was directed to mass detector in gas chromatograph system,

while the other part was directed to an olfactory detection port (ODP). Humidified air

was introduced into the sniff port up-stream at 100 mL/min near the point where the

capillary column first entered the sniffing port. The air carried the capillary column

effluent into a glass funnel where sensory analysis was done. The transfer line to the

GC-O sniffing port was held at 280 °C. Helium was introduced as make up gas in the

transfer line to the sniffing port at 8 mL/min. Simultaneous sniffing at the exit port of

ODP and corresponding response in the mass detector allowed identification of the

compound responsible for the perceived aroma based on its mass fragmentation. The

operating conditions were similar to that provided in section 2.10.1.2. Peaks

corresponding to various aroma notes sniffed by the assessors were identified by

comparing their mass fragmentation pattern with that of standard spectra available in the

spectral library (Wiley/NIST Libraries) of the instrument as well as based on their

87

retention indices on the DB-5 column previously reported, odor quality and mass

spectral data with those of standard compounds wherever available.

2.2.2.2.4.2. Global Olfactometric analysis and determination of odor activity values

(OAV)

Analysis was carried according to the conditions reported by Guen et al. [135]. A panel

of 9 judges (4 females and 5 males, aged 25-50 years) trained in odor recognition and

experienced in GC-O was selected. The testing room was at 24 ± 1 °C and 50 ± 5% RH;

the illumination was a combination of natural and non-natural (fluorescent) light.

Panelists assigned odor properties to each note detected. The number of times a note

was perceived by sensory panel was defined as its detection frequency. Detection of

odor by fewer than 4 panelists was considered as noise [136]. Sniffing was divided into

two parts each of 15 min. Panelists were divided in two batches consisting of four and

five people. Panelists involved in first 15 min were asked to sniff another half of the

sniffing in next round. The second batch sniffed the remaining 15 min of the first round.

In the second round of analysis, first batch of panelists analyzed the aroma notes eluting

from 15 to 30 min while the second batch analyzed the aroma compounds eluting during

0-15 min. Thus, each person participated in the sniffing of both parts but during two

distinct sessions to avoid tiredness. A final aromagram of detection frequency (DF)

versus RI was obtained after summing up all the nine individual aromagrams.

To evaluate the contribution of a chemical compound to the overall aroma of a

vegetable the odor activity value (OAV) was determined. OAV is a measure of

importance of a specific compound to the odor of a sample. It was calculated as the ratio

88

between the concentration of an individual compound and the perception threshold as

reported in literature [137].

2.2.2.3. Headspace gas composition inside packages

Oxygen concentration in package headspace was measured using a gas chromatograph

(GC 2010, Shimadzu Corporation, Japan). The GC was equipped with split/splitless

injector, a molecular sieve column (length 30 m, 0.35 I.D., RT-Msieve 5A, Restek

Corporation, USA) and a TCD detector. Injection port temperature was 35 °C. Initial

column temperature was kept 30 °C for 5 min and then raised at rate of 0.167 °C/s to

100 °C. The column was further held at 100 °C for 5 min with the TCD current and

temperature maintained at 90 mA and 110 °C, respectively. Sampling was done by

inserting a hypodermic needle into the bag through an adhesive septum, previously

stuck to the bag. A 0.1 mL of headspace sample was extracted and injected into a split

ratio of 5. Only O2 and N2 could be evaluated on the column used in the study. Based on

observed O2 and N2 concentrations in the package headspace, actual concentrations of

O2 and CO2 (% O2 and % CO2) were calculated using following equations:

% O2 = (Observed % O2) × 78.084 (1)

(Observed%N2)

% CO2 = 100 – (Observed % O2) × 78.084 + 78.084 (2)

(Observed % N2)

(The atmospheric composition of N2 was taken as 78.084%).

Analysis was done in triplicates using three independent sample trays on each day.

2.2.2.4. Gamma irradiation induced browning inhibition

2.2.2.4.1. Isolation and identification of phenolic compounds

89

Isolation of phenolic compounds: Ash gourd (20 g) from each tray was extracted twice

with 20 ml methanol. The extract was then centrifuged at 12000 rpm for 20 min and the

supernatant collected. It was filtered through 0.22 µm membrane filters (Millipore) prior

to analysis by HPLC.

HPLC analysis: Phenolic constituents were evaluated by a reversed-phase high-

performance liquid chromatography (RP-HPLC, Jasco, Tokyo, Japan), equipped with a

degasser, a quaternary pump delivery system, and a diode array detector. Sample (20

µL) as obtained above was loaded on to a C-18 column (250 mm × 4.6 mm length, 5 μm

particle size) and eluted with mobile phase (flow rate 1 mL/min) consisting of solvent

A- 1.5% o-phosphoric acid in Milli Q water and solvent B- acetonitrile: acetic acid:

phosphoric acid: water (20:24:1.5:54.5). The gradient program used was as follows: 0

min- 80% A; 30 min- 33% A; 33min- 10% A; 40 min – 0% A. The column was

conditioned with acetonitrile for 10 min after every run. The peaks were identified by

comparing retention times and UV-Vis spectra in the 200-500 nm range with those of

the corresponding authentic standards. Quantification of identified compounds was

performed using calibration curves of the corresponding standard compounds at the

specific absorption maximum. Co-chromatography with added authentic standards was

also performed for further confirmation of the identified phenolic constituents.

2.2.2.4.2. Effect of radiation processing on enzymes involved in browning

Phenyl alanine lyase (PAL): PAL activity was measured according to the protocol

reported by Degl’Innocenti et al with some modifications [138]. Enzyme extracts were

obtained by blending 1:1 (w/w) cut ash gourd pieces with borate buffer (50 mM, pH 8.5)

90

containing 10 mM 2-mercaptoethanol and 0.5 g of PVPP under chilling conditions. The

homogenate was filtered through 4 layers of cheesecloth and centrifuged at 21,000 x g at

4 °C for 20 min. The supernatant was used for subsequent assay. The reaction mixture

consisting of 1 mL of 50 mM L-phenylalanine, 0.5 mL of the supernatant and 1.5 mL of

borate buffer was incubated at 40 °C for 1 h. The absorbance was measured at 290 nm

before and after incubation. Difference between the two gave the amount of product

(cinnamic acid) formed. One unit of PAL activity equals the amount of PAL that

produced 1 μmol of trans-cinnamic acid in 1 h and was expressed as μmol g-1

FW h-1

.

Peroxidase (POD): The enzyme extract was by blending 1:1 (w/w) cut ash gourd pieces

with 0.05 M phosphate buffer at pH 7.0 under chilling conditions. Blending was

performed in the presence of 10 % (w/w) polyvinylpyrrolidone (Sigma, St. Louis, MO,

USA) to bind polyphenols. The extract was then centrifuged at 4 °C at 21,000 x g for 10

min. The supernatant was collected and used for peroxidase (POD) activity. Assay was

carried according to the procedure reported by Degl’Innocenti et al (2005) with some

modifications 138. The chlorogenic acid peroxidase assay contained 750 μL of 50 mM

potassium phosphate buffer, pH 6.5; 100 μL of 80 mM chlorogenic acid; 50 μL of

extract, and 100 μL of 35 mM H2O2. The caffeic acid peroxidase assay contained 750

μL of McIlvaine buffer (114 mM Na2HPO4 and 43 mM citric acid), pH 5.5; 100 μL of

80 mM caffeic acid, 50 μL of extract; and 100 μL of 35 mM H2O2. In all cases, POD

assays were initiated by the addition of H2O2 (100 μL, 35 mM). Absorbance was

measured at 410 nm for chlorogenic acid and 470 nm for caffeic acid peroxidase

activity. The activities of PODs were expressed as Aλ min-1

g-1

fresh weight.

91

Polyphenol oxidase (PPO): Enzyme extraction procedure followed was same as that

for POD assay as mentioned above. PPO activity was assayed spectrophotometrically

(Model UV 4-100, Unicam, Cambridge, UK) at 25 °C according to methodology of

Mishra et al. [139] based on the absorption at 420 nm of the brown polymers formed

when catechol is oxidized in the presence of PPO. The reaction mixture consisted of 600

µL buffer, 200 µL enzyme extract and 200 µL substrate. The increase in absorbance at

420 nm was monitored at 1 min intervals for 10 min and the average change in

absorbance per minute was calculated from the linear region of absorbance. One unit of

enzyme activity was defined as the amount of enzyme which caused a change of 0.01 in

absorbance/min. The PPO activity was expressed as U g-1

fresh weight.

PPO inhibition – Kinetic studies: The inhibitory activity of alpha resorcylic acid for

ash gourd PPO was investigated. Various concentrations of catechol (25 - 250 mM) and

alpha resorcylic acid (50 - 250 mM) were prepared in phosphate buffer (pH 7). Catechol

solutions (150 µL) and alpha resorcylic acid (100 µL) were mixed together in a cuvette

containing 550 µL of the buffer. The reaction was initiated by addition of 200 µL of

PPO preparation. The readings were taken in the similar manner as described above

(section 2.11.1.1.4.1(C)). The degree of inhibition was expressed as percentage

inhibition (I = 100(A* - A)/A, where A and A* were enzyme activities with or without

inhibitor, respectively). Kinetic parameters of alpha resorcylic acid inhibition were

determined using double-reciprocal plots (Lineweaver-Burk) of enzyme activity vs

catechol concentration. I50 value of alpha resorcylic acid was determined from the plot

92

between enzyme activity vs inhibitor concentration, where I50 value is the mM

concentration of the inhibitor required to inhibit enzyme activity by 50 %.

2.2.2.4.3. Scanning Electron Microscopic (SEM) analysis

Control and irradiated ash gourd pieces on day 12 of storage were subjected to SEM

analysis using a facility (SEM Model Quanta 200 SEM, FEI Company, Oregon, USA)

available from Icon Analytical Equipments Pvt. Ltd., Mumbai, India. The sample was

fixed on a carbon tape and observed at different magnifications using a low vacuum

mode at 65 Pa and 20 kV.

2.2.2.4.4. Electrolytic leaching

The electrolytic leaching was measured using the procedure as reported earlier. 10 g of

cut ash gourd was taken in a beaker containing 50 ml of deionized water and stirred on a

magnetic stirrer for 2 minutes. It was then filtered through Whatman filter paper and

conductance of the filtrate was measured using a hand-held conductivity meter (HI

9812, Hanna Instruments, Italy). Spectrophotometer readings of the filtrate were also

taken at 280, 320 and 420 nm for assessment of nature and content of compounds

leaching out and hence attributing to the browning.

2.2.2.4.5. Statistical analysis

Statistical significance was assessed by two-way analysis of variance to determine the

effect of radiation processing and storage time. Multiple means comparison was done by

Duncan’s test (p<0.05). For each measurement, three replicates of 3 independent

samples were tested.

93

2.2.3. Isolation, identification and studying the effect of radiation processing on

bioactive phytochemicals

(A) Ash gourd

2.2.3.1. Compounds with plant growth promoting activities

2.2.3.1.1. Preparation of ash gourd extracts

Ash gourd (5 kg) was cut in small pieces and soaked in aqueous methanol (1:4 v/v)

overnight. The supernatant was removed by filtration through a sintered funnel under

vacuum and the remaining ash gourd was crushed in an omni mixer for 2 min at a speed

corresponding to position of the knob at 3. The slurry was filtered as above and the

residue was further re-extracted twice with aqueous methanol. The filtrates were pooled

and concentrated under vacuum on a rotary evaporator (35 °C) to obtain an aqueous

solution.

The aqueous solution so obtained (500 ml) was filtered and then extracted with n-

hexane (3 x 250 ml) in a separating funnel. The organic layers were pooled, washed

with water (2 x 250 ml) and then evaporated to dryness under vacuum in a flash

evaporator. The residue was made to 1% solution in DMSO. The remaining aqueous

solution was consecutively extracted with ethyl acetate (3 x 250 ml) and n-butanol (3x

250 ml) as above. The organic layers in each case as well as the remaining aqueous

solution were separately evaporated to dryness as above and made to 1% solution in

ethanol. These fractions were tested for their growth promoting activity.

94

2.2.3.1.2. Activity Guided Fractionation

2.2.3.1.2.1. Growth promotion activity: Growth promotion activity was examined

using tobacco plant because of its extensive and well characterized genetic background.

Tobacco leaves were cut into circular discs of diameter 0.5 cm with the help of a sterile

metallic cork borer. Discs obtained from the middle regions (between midrib and edge)

of tobacco leaves were treated by soaking them overnight in MS liquid medium

containing different concentrations of all the above extracts (0.178 – 4.4 µg/ml). Leaf

discs soaked overnight in liquid media were used as control. The flasks were kept

overnight on an orbital shaker at 100 rpm and maintained at 25 °C. The treated leaf discs

were transferred to petri plates having half strength MS media containing 0.2 % agar, 3

% sucrose and 0.04% calcium chloride, adjusted to pH 5.7. The plates were then sealed

with parafilm and kept in growth chambers under a 16 h photoperiod at 25 ± 1 °C for 21

d. The diameter of the incubated leaf discs were measured on day 3, 7, 14 and 21.

Except for the total aqueous extract and the butanol extract, no increase in leaf disc

diameter was observed with other fractions. The total aqueous extract as well as the

butanol extract exhibited similar activity with an increase in diameter of tobacco leaf

discs up to 1.1 cm on day 14. The leaves were found to undergo senescence beyond the

period of 14 days.

2.2.3.1.2.2. TLC analysis

The butanol fraction was subjected to thin layer chromatography. Analytical TLC was

carried out on ammonium sulphate impregnated silica gel G plate (10 cm x 25 cm x 0.25

mm thickness) using toluene:ethanol:formic acid (60:35:5, v/v/v) as the developing

95

solvent system. The separated spots were visualized by heating the plate in an oven at

120 °C for 20 min. The butanol extract was subsequently purified by preparative thin

layer chromatography. Preparative TLC was carried out on silica gel plates (20 cm x 20

cm x 0.5 mm thickness) using the same solvent system as above. Five distinctly

resolved bands were scrapped and eluted with methanol. The eluate was evaporated to

dryness to make 1% solution (w/v).

2.2.3.1.2.3. Estimation by densitometry

Density of the individual spots/ bands was determined on a dual wavelength flying spot

scanning densitometer Shimadzu CS-9301PC (Shimadzu, Kyoto, Japan). The density of

the spots was determined in the reflectance mode at a wavelength of 528 nm. Acetoin

glucoside was used as external standard. Aliquots of suitably diluted sample were

spotted on the plate in increasing concentration ranging from 0.05 to 6 mg/ml. The

amount of different bands was estimated from a standard curve (R2 = 0.99) of spot

density versus concentration and expressed as mg kg-1

of ash gourd.

2.2.3.1.2.4. Determination of active principles

The bands at Rf values 0.38, 0.44, 0.52, 0.59 and 0.77 were scrapped from the plate and

eluted separately with distilled ethanol. Each solution was filtered, concentrated and

made to 1% solution in ethanol. These fractions were tested for their growth promoting

activity in order to identify the most active fraction. The growth promoting activity

mainly resided in the band at Rf 0.44 which showed an increase in diameter of leaf discs

up to 1.13 cm as compared to control (0.58 cm) on day 14. However, no increase in leaf

disc diameter was observed when treated with the other four bands. Identification of the

96

active compound present in the TLC band at Rf 0.44 was further carried out as

described below.

2.2.3.1.3. Structure identification

The preliminary identification of the compound present in the TLC band at Rf 0.44 was

performed by GC/MS after acidic hydrolysis with the procedure as discussed earlier

(section 2.10.2.3). The compound was tentatively identified as acetoin glucoside. The

remaining aqueous solution from the individual bands was neutralized with 1 N KOH,

dried under vacuum and the residue was dissolved in methanol. This methanol solution

was subjected to TLC in order to identify the sugar residue. The sugar residue obtained

from the hydrolyzate from the individual bands was further converted to its acetyl

derivative using pyridine : acetic anhydride (1:1) (overnight, room temperature). The

acetylated derivative was concentrated by slow stream of nitrogen and then

subsequently analyzed by GC/MS.

Identity of the active compound present in this band was substantiated by other spectral

techniques such as IR, NMR and further by chemical synthesis.

2.2.3.1.3.1. Spectral Studies

IR spectrum was recorded with a JASCO FTIR 4100 spectrophotometer (Jasco

Corporation, Tokyo, Japan). νmax (KBr): 3520, 2980, 1665, 1452, 1385, 1180, 1114

and 910 cm-1

.

The NMR spectra were recorded with a Bruker AC-200 MHz FT NMR spectrometer

(Bruker, Fallanden, Switzerland). The usual abbreviations employed are: s = singlet, d =

doublet, q = quartet, J = coupling constant (in Hz), δ = chemical shift in ppm. The

97

proton designations as per chemical structure depicted in Fig. 10. 1H NMR (CDCl3, 200

MHz): δ ppm, 4.5-5.2 (sugar protons, H2’, H3’, H4’, H5’ & H6’), 4.22 (1H, q, H3, J =

5.4), 4.2 (1H, d, J = 5.8, H1’), 2.01 (3H, s, H1), 1.30 (3H, d, J = 5.4, H4).

2.2.3.1.3.2. Chemical synthesis of acetoin glucoside

Further confirmation of the structure was carried out by chemical synthesis according to

the scheme depicted in Figure 11. In a 3- necked round bottom reaction flask,

continuously flushed with argon, a mixture of sodium hydride in dry THF was added.

Acetoin (88 mg) was then introduced into the flask in a controlled manner by using

pressure equalizer. The reaction mixture was refluxed for 1h until the added acetoin was

converted to its corresponding sodium derivative and effervescence of hydrogen had

stopped. Acetobromo-D-glucose (397 mg) in THF was then added drop wise with the

help of pressure equalizer with continuous stirring at room temperature for the

formation of glycosidic bond between glucose and sodium derivative of acetoin. The

reaction product was purified by column chromatography using binary solvent

combination (15% ethyl acetate in petroleum ether) followed by preparative TLC using

ethyl acetate: petroleum ether (15:85, v/v) as developing solvent. The final purified

product was then characterized by spectral analysis as above.

Figure 11. Scheme of chemical synthesis of acetoin-3-O-β-D- glucoside.

OOAc

OAc

AcO

BrOH

O + (iii). Stirring at rt for over night and worked up

(i). NaH in freshly dry THF (ii). Refluxed for 1h

AcO

O

OOAc

OAc

AcO

O

AcO

1

23

1'2'

3'

4'5'

6'1

23

1'2'

3'

4'5'

6'

4

4 Acetoin Acetobromo-D-glucose Acetoin-3-O- β-D-glucoside

98

2.2.3.1.4. Growth promotion assay of purified acetoin glucoside

Tobacco leaf discs were treated with synthetic acetoin glucoside and that isolated from

ash gourd, as well as standard acetoin procured from Sigma Aldrich, at concentrations

ranging from 0.178 – 4.4 µg/ml. Leaf discs were cultured in similar growth conditions

as mentioned above. The growth of the leaf discs was monitored at different time

intervals (3, 7, 14 and 21 days). The growth observed was identical in both natural and

synthetic acetoin glucoside. Therefore glucoside obtained from the natural source was

used in subsequent studies. Three independent sets of experiments were performed and

the data presented as a mean ± standard deviation.

2.2.3.1.5. Protein extraction and two-dimensional gel electrophoresis

Proteins were extracted using 0.1 M phosphate buffer supplemented with 0.1% Triton-X

100, 20 mM EDTA, 0.1 M NaCl, 5 mg/ml sodium ascorbate and 2 mM PMSF. Control

and treated in vitro grown plant tissues (1 g fresh weight) were ground with liquid

nitrogen using sterilized mortar and pestle. The powder was suspended in 1 ml of the

above buffer. After incubating for 2 h at 4˚C, the samples were centrifuged at 12,000 g

at 4˚C for 30 min. The centrifugation was repeated with the supernatant. The final

supernatant was collected and equal amount of protein (90 µg) was used for iso-electric

focusing (IEF). 2-D gel electrophoresis and IEF was done using an 11 cm strip as

reported earlier [140] with the following focusing conditions- 250 V, 15 min (rapid, salt

removal step); 8000 V, 2h (linear); 8000 V, 20-30,000V-h (rapid) and finally 500

V, hold. 2-D separation was done on a 12% PAGE gel and was silver stained.

99

2.2.3.1.6. Mass Spectrometric analysis and database searching

Spots were excised manually from gels and digested with trypsin. The digestion

protocol used was as per the procedure reported earlier [141] with minor modifications.

Gel plugs were initially destained with 20 mM ammonium bicarbonate. Trypsin (250

ng), at a concentration 25 ng/μl, was then added and the digestion proceeded at 37°C for

16 hrs. Peptides were extracted from gel plugs by adding 0.1% TFA containing 50%

ACN/Water. Peptide fragments from digested proteins were then crystallized with

saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix solution. The MS analysis

was performed in 4800 MALDI-TOF/TOF (Applied Biosystems, Foster City; CA) mass

spectrometer in the m/z range 600-4500 Da. Peptide mass fingerprinting (PMF) search

was performed using Swissprot database, using the MASCOT search engine (Matrix

Science Ltd., London; http:/www.matrixscience.com).

2.2.3.1.7. Statistical Analysis

Experimental data of growth promotion studies were analyzed statistically by two-way

ANOVA using DSAASTAT (Version 1.1) [142] to determine the effect of various

treatments at different concentrations during the observed growth period. Multiple

means comparison was done by Duncan's multiple range test (p≤0.05).

2.2.3.2. Compounds with ACE inhibition activity

2.2.3.2.1. Preparation of ash gourd extracts

The preparation of ash gourd extract and its subsequent partitioning with n-hexane, ethyl

acetate and n-butanol was performed as explained in section 2.2.3.1.1. The organic

100

layers in each case as well as the remaining aqueous solution were separately evaporated

to dryness and made to an aqueous solution (100 mg/ml). These fractions were tested for

ACE inhibition activity.

2.2.3.2.2. Preparation of enzyme extract

Angiotensin converting enzyme (ACE) was extracted from pig (Sus scrofa) lung tissue.

10 g pig (Sus scrofa) lung tissue was minced using scissors on an ice bath and made to a

volume of 100 mL with 10 mM sodium phosphate buffer (pH 8.3). The mixture was

homogenized in chilled conditions. The homogenate was centrifuged at 21000 x g, for

20 min at 4 °C. The supernatant obtained was used as enzyme extract and stored at -20

°C till the analysis.

2.2.3.2.3. ACE inhibition assay

The ACE inhibition activity was measured using the procedure reported by Cushman

and Cheung [143] with slight modifications. 50 µL of enzyme preparation was mixed

with 50 µL of inhibitor (ash gourd extract) and pre-incubated at room temperature for 10

min. The mixture was designated as “test”. A mixture of 50 μL distilled water + 50 μL

enzyme was taken as control. 250 µL of N-Hippuryl-His-Leu tetrahydrate (HHL) as

substrate (prepared in HEPES buffer) was added to the pre-incubated test and control

mixture and incubated at 37 °C for 1 h. After 1 h, the reaction was stopped by adding

200 μL of 1 N HCL in each tube. 1 mL of ethyl acetate was then added and mixed

thoroughly by vortexing for 90 s. They were then centrifuged at 21000 x g for 10 min.

700 μL aliquot from supernatant of each tube was collected in separate tubes and

evaporated to dryness in a boiling water bath. After complete drying, 1 mL distilled

101

water was added in each tube and OD of aqueous solutions so obtained was read at 228

nm using UV spectrophotometer (Helios α). The assay was performed in triplicate for

both sample and control tubes. The % inhibition activity was calculated as: [(ODC -

ODT)/ ODC]*100. The ACE inhibition activity mainly resided in the aqueous extract

obtained after hexane, ethyl acetate and butanol fractionation. This aqueous extract was

therefore further fractionated for determining the active principles.

2.2.3.2.4. Activity guided fractionation and identification of active principles

HPLC and GPC: The aqueous extract after fractionation with organic solvents as

above was injected in RP-HPLC equipped with a hypercarb C18 column (150 mm x 4.6

mm length, 5 µm particle). The solvent system employed for elution consisted of a

gradient of 0.1 % TFA and acetonitrile at a flow rate of 1 mL/min. The peaks were

monitored at 220 nm. The major peaks observed were collected in separate tubes and

lyophilized. The dry residue was then reconstituted in appropriate volume of distilled

water and checked for ACE inhibition activity. The activity mainly resided in a peak at

retention time 2.9 min. This peak was further fractionated using gel permeation

chromatography with a SuperdexTM

peptide 10/300 GL column. Out of several collected

peaks from the column, the activity was present mainly in a peak at retention time of 30

min. This peak was repeatedly purified using GPC and subjected to TLC and GC/MS

analysis.

TLC analysis: The aqueous extract obtained after partitioning with organic solvents and

the purified peak obtained from GPC as above was spotted on an analytical TLC plate

using toluene: ethanol: formic acid (6: 3.5: 0.5) as the developing solvent. After

102

development, the plate was dried at room temperature and then sprayed with 0.5 %

ninhydrin solution in acetone for visualization.

GC/MS: The purified peak was subjected to mass spectroscopy by using direct probe

facility available with the GC/MS (Schimadzu GC-17A). The active principles were

thus tentatively identified by analyzing the mass spectral data obtained.

(B) Pumpkin

2.2.3.3. Isolation and identification of phenolic constituents

The methodologies adopted for isolation and identification of major phenolic

compounds in pumpkin were similar to that as described in section 2.2.3.1.

2.2.3.4. Isolation and identification of carotenoids

2.2.3.4.1. Isolation of carotenoids

Ten grams of pumpkin from each tray was extracted twice with 10 mL hexane

containing 10 mg of butylated hydroxytoluene (BHT). The extract was then centrifuged

(16000 x g, 20 min, 4 °C) and the supernatant was collected. It was then passed through

the anhydrous sodium sulphate. The extract so obtained was evaporated to dryness

under vacuum (T < 30 °C) and the residue was made to 0.05% in a solvent mixture

consisting of acetonitrile: tetrahydro furan (THF): methanol: 1% aq. ammonium acetate

solution: BHT (55:35:5:5:0.05, v/v/v/v/w) as reported by Seo et al. [144]. Finally the

solutions were filtered through 0.22 µm membrane filters (Millipore) prior to injection

(20 µl) into the HPLC.

103

2.2.3.4.2. HPLC analysis

Carotenoids were evaluated by reversed-phase high-performance liquid chromatography

(RP-HPLC, Jasco, Tokyo, Japan), equipped with a degasser, a quaternary pump delivery

system, and a diode array detector. Separations were conducted using C-18 column (250

mm × 4.6 mm length, 5 μm particle size) with a solvent flow rate of 1 ml/min. The

solvent system used for carotenoid analysis consisted of solvent A- acetonitrile: THF:

methanol: 1% aqueous ammonium acetate solution: BHT (85:5:5:5:0.05, v/v/v/v/w) and

solvent B- acetonitrile: THF: methanol: 1% aq ammonium acetate solution: BHT

(55:35:5:5:0.05, v/v/v/v/w) [144]. The gradient employed was as follows: 0-10 min, 5%

B; 10-29 min increasing linearly to 95% B; 29-35.9 min, maintaining 95% B; decreasing

to 60 % B at 36 min; maintaining 60% B from 36-44.9 min. Carotenoids were

monitored at 452 nm. The peaks were identified by comparing retention times and UV-

Vis spectra in the 200-500 nm range with those of the corresponding authentic

standards. Quantification of identified compounds was performed using calibration

curves of the corresponding standard compounds at the specific absorption maximum.

Co-chromatography with added authentic standards was also performed for further

confirmation of the identified carotenoids.

2.2.3.5. Isolation and identification of triterpenes

2.2.3.5.1. Isolation of triterpenes

Pumpkin sample (10 g) from each tray was extracted twice with 20 mL methanol using

a high speed omni mixer. The homogenate obtained was filtered through double layered

washed and dried muslin cloth. The extract obtained was centrifuged (12,000 rpm, 20

104

°C, 15 min) and solution thus obtained was evaporated to dryness under vacuum (40

mbar, 40 °C) using a rotary evaporator (Buchi, Switzerland). The dried residue was

made to 1% solution in methanol.

2.2.3.5.2. Identification of bioactive triterpenes

TLC analysis: The methanol extracts obtained as above were spotted on an analytical

TLC (silica G, 0.25 mm) plate using toluene: ethanol: formic acid (5:4:1) as the

developing solvent system. The developed plates were dried at room temperature and

stained with iodine vapors. The spots so obtained were scanned on a Flying Spot

Scanning Densitometer (CS9301PC, Shimadzu, Japan) and the area of each spot was

then estimated. After the iodine vapors faded away, the plate was sprayed with

Liebermann-Burchard reagent (5 mL acetic anhydride + 5 mL conc. H2SO4 + 50 mL

ethanol), and visualized by heating the plate in oven at 120 °C for 20 min. The methanol

extract was concentrated to dryness using rotary evaporator and hydrolyzed by pectinase

enzyme (section 2.2.2.2.2.3.) The hydrolyzate along with the original methanol extract

was again subjected to analytical TLC with different solvent systems (1- ethyl acetate :

benzene (75:25) ; 2- chloroform : acetone (90:10)) to determine the nature of the

compounds. Tentative identification of the resolved bands was performed by matching

their Rf values available in literature.

HPLC analysis: The methanol extract as obtained above was subjected to reverse-phase

high-performance liquid chromatography (RP-HPLC, Jasco, Tokyo, Japan), equipped

with a degasser, a quaternary pump delivery system, and a diode array detector.

Separations were conducted using C-18 column (250 mm × 4.6 mm length, 5 μm

105

particle size) with a solvent flow rate of 1 ml/min. An isocratic solvent system

consisting of methanol: water (2:1) was used for elution. The peaks were monitored at

237 and 254 nm by a photo diode array detector operating in a wavelength range of 220-

400 nm. Tentative identification of the peaks was performed on the basis of their

retention times available in the literature.

GC/MS analysis: In order to further ascertain the identities of the active principles, the

hydrolyzate of the total extract, as obtained above was analyzed by GC/MS.

Identification was carried out by analyzing the mass spectral data thus obtained.

(C) Drumstick

2.2.3.6. Isolation, identification and quantification of phenolic constituents

The methodologies adopted for isolation and identification of major phenolic

compounds in drumstick were similar to that as described in section 2.2.3.1.

2.2.3.7. Isolation and identification of glucosinolates

50 g of cut drumstick from each tray was taken in 85 ml of boiling water and subjected

to microwave treatment for 3 min to inactivate the inherent myrosinase enzyme present

in the vegetable. The mixture was then homogenized using a high speed homogenizer

(B400, Buchi, Switzerland) and the slurry was filtered through double layered washed &

dried muslin cloth. The extract obtained was centrifuged at 21000 x g at 20 °C for 15

min and subjected to HPLC and GC/MS analysis as briefly described below.

106

HPLC analysis: The aqueous glucosinolate extract was subjected to HPLC (Jasco

HPLC system, Japan). Samples were eluted from a reverse phase C18 column (250 mm

x 4.6 mm, 10 μ; HYPERSIL, Chromato-pack, Mumbai, India) using 0.1 % TFA in

water as solvent A and 0.1 % TFA in methanol as solvent B. The solvent gradient

employed was: At t = 0 - 5 min, A= 100%; t = 15-17 min, A = 83%; t=22 min, A = 75

%; t=30 min, A= 65%; t=35 min, A=50%, t=50 min, A=1%; t=55-60 min, A=100%, at a

flow rate of 1.0 mL/min. Wavelength was set at 227 nm.

Glucosinolates were desulfated using 10 mL crude aqueous extract (10% solution) to

which 500 μl of 0.02 M sulfatase enzyme in aq. NaOAc-AcOH (pH 5) was added and

incubated overnight [145]. The resultant mixture was subjected to HPLC analysis as

above for further confirmation of glucosinolates.

GC/MS analysis: 15 ml of the crude aq extract was evaporated to dryness in rotavapor

and then reconstituted in 15 mL of 0.1 M phosphate buffer (pH 7.0). It was then added

with 2 mg of myrosinase (Sigma) and incubated at 37 °C for 3 h. After completion of

hydrolysis, samples were equilibrated for 45 min at 30 °C with continuous shaking and

headspace was subsequently extracted by preconditioned PDMS/DVB/CAR fibres

(Supleco, USA) for 20 min at 30 °C. After extraction, fibres were desorbed (270 °C) in

injection port of GC/MS. 2-octanol (1.25 μg) was used as internal standard for

quantification. Peak areas of compounds were estimated by comparing with those of

internal standard and results reported as μg kg-1

.

107

CHAPTER 3

RESULTS AND DISCUSSION

108

3.1. Development of radiation processed shelf stable RTC vegetables

Five popular Indian vegetables - ash gourd (Benincasa hispida), pumpkin (Cucurbita

pepo), bottle gourd (Lagenaria siceraria), lady finger (Abelmoschus esculentus) and

drumstick (Moringa oleifera) were selected for the preliminary studies. Besides their

nutritive value, these vegetables have also been used traditionally for their

pharmacological and medicinal value. All the five vegetables were processed as described

in materials and methods (section 2.2.1.) and stored at 10 °C. The samples were

monitored at suitable intervals visually as well as by the sensory panel for appearance,

color, texture and aroma. In the case of ready-to-cook (RTC) ash gourd, pumpkin and

drumstick, radiation processed samples were acceptable up to a storage duration of 10-25

d depending on the vegetable and radiation dose delivered. In case of bottle gourd, the cut

vegetable (control as well as radiation processed) developed browning / blackening after a

few minutes of cutting, making them unacceptable. In case of ladies finger, the treated

and untreated samples were acceptable only up to a storage period of 1-2 d, beyond which

increased sliminess and off odor in the radiation treated samples restricting their

acceptance by the sensory panel. Preliminary investigations thus showed that radiation

processing was not amenable for the shelf life extension of RTC bottle gourd and lady

finger. Hence ash gourd, pumpkin and drumstick were selected for the further studies.

3.1.1. Optimization of radiation dose and storage period with mathematical

modeling

The shelf-life of fresh-cut fruits and vegetables depends on various factors such as

processing conditions, treatment, storage temperature, packaging conditions, microbial

109

and physiological spoilage, that affect visual appearance, firmness and consumer

acceptance. Understanding the response of a food system to qualitative or quantitative

experimental variables is a complex task. Efficiency of mathematical modeling to

optimize the processing conditions based on various experimental designs has been

widely demonstrated [146]. Such mathematical equations can be utilized in the food

industry for deciding the process parameters required for obtaining the desired shelf-life

of fresh-cut produce, thus reducing severity of treatments.

In the present case, full factorial experimental design approach was employed for

optimization of radiation dose and storage time for obtaining RTC products of the

selected vegetables with acceptable microbial and sensory quality. A total of 90

experiments were performed for each vegetable at six radiation doses (0 – 2.5 kGy, at an

interval of 0.5 kGy) and five storage days (0, 5, 8, 12 and 15 for ash gourd and drumstick;

0, 7, 14, 21 and 28 for pumpkin). The samples at each combination of dose and storage

day were analyzed in triplicates for microbial quality (Total plate count (TPC) and Yeast

& mold count (Y&MC)), color, texture and sensory acceptability as per procedure

explained in materials and methods (section 2.2.1.). The experimental design and data

obtained for ash gourd, drumstick and pumpkin is presented in Tables 17, 18 and 19,

respectively.

The data obtained for each quality parameter (TPC, Y&MC, color, texture and overall

acceptability) was individually fitted in cubic polynomial equations and models were

generated. These polynomial equations were used to explain the complex interactions

among radiation dose, storage duration and quality attributes of the RTC products.

110

Statistical parameters of the models generated for ash gourd, drumstick and pumpkin are

represented in Tables 20, 21 and 22, respectively. Statistical analysis by ANOVA showed

that the mathematical models generated for each product were significant (p < 0.05),

while lack of fit was insignificant (p > 0.05). Further, the signal to noise ratio (S/N) for all

models were above 4 indicating sufficient data to navigate designed space. Coefficients

for predicted regression models and coefficient of determination (R2) are shown in Tables

23, 24 and 25 for ash gourd, drumstick and pumpkin respectively. Good fitting of models

is also indicated by high values of R2.

Table 17. Experimental design for RTC ash gourd

Std Run Factor 1 A:Dose (kGy)

Factor 2 B: Storage duration (d)

TPC (Log CFU/g)

Y & MC (Log CFU/g)

L value

Firmness (N)

Overall acceptability

1 1 0(-1) 0(-1) 3.87 2.09 68.41 3.475 7.7 2 2 0(-1) 0(-1) 3.79 2.16 68.31 3.584 7.6 3 3 0(-1) 0(-1) 3.92 2.05 69.65 3.506 7.8 4 4 0.5(-0.6) 0(-1) 3.4 1.37 68.43 3.096 7.5 5 5 0.5(-0.6) 0(-1) 3.27 1.25 67.88 2.994 7.7 6 6 0.5(-0.6) 0(-1) 3.22 1.22 67.54 2.905 7.3 7 7 1(-0.2) 0(-1) 2.62 1 66.6 3.094 7.5 8 8 1(-0.2) 0(-1) 2.65 1 65.21 2.996 7.6 9 9 1(-0.2) 0(-1) 2.54 1 64.96 3.154 7.4 10 10 1.5(0.2) 0(-1) 2.63 1 68.54 2.844 7.3 11 11 1.5(0.2) 0(-1) 2.54 1 66.58 2.965 7.5 12 12 1.5(0.2) 0(-1) 2.54 1 67.45 2.881 7.1 13 13 2(0.6) 0(-1) 2 0.65 65.99 2.359 6.8 14 14 2(0.6) 0(-1) 1.85 0.7 64.69 2.296 7.1 15 15 2(0.6) 0(-1) 1.75 0.8 66.21 2.408 6.5 16 16 2.5(1) 0(-1) 1.06 0.5 66.65 2.5 5.2 17 17 2.5(1) 0(-1) 0.96 0.5 63.32 2.25 5.6 18 18 2.5(1) 0(-1) 1.11 0.5 67.12 2.21 4.8 19 19 0(-1) 5(-0.33) 8.71 2.07 63.52 2.475 1.9 20 20 0(-1) 5(-0.33) 8.54 1.95 62.54 2.562 2.1 21 21 0(-1) 5(-0.33) 8.13 2.06 61.38 2.333 1.7 22 22 0.5(-0.6) 5(-0.33) 6.38 2.04 64.71 2.839 3.1 23 23 0.5(-0.6) 5(-0.33) 6.58 2.15 64.17 2.882 3.3 24 24 0.5(-0.6) 5(-0.33) 5.85 1.95 63.22 2.915 2.9 25 25 1(-0.2) 5(-0.33) 5.97 2.15 67.96 2.889 5.4 26 26 1(-0.2) 5(-0.33) 5.56 1.96 67.55 2.914 5.6 27 27 1(-0.2) 5(-0.33) 5.03 2.2 66.95 2.814 5.2 28 28 1.5(0.2) 5(-0.33) 4.05 1.85 67.2 2.787 4.3 29 29 1.5(0.2) 5(-0.33) 3.95 1.75 67.55 2.669 4.4 30 30 1.5(0.2) 5(-0.33) 4.15 1.88 66.19 2.751 4.2 31 31 2(0.6) 5(-0.33) 2.16 1 61.73 2.354 7.1 32 32 2(0.6) 5(-0.33) 2.05 1 62.52 2.145 7.2 33 33 2(0.6) 5(-0.33) 2.2 1 63.51 2.262 7 34 34 2.5(1) 5(-0.33) 2.38 1 62.82 2.063 5.6 35 35 2.5(1) 5(-0.33) 2.35 1 63.98 1.996 6.2 36 36 2.5(1) 5(-0.33) 2.3 1 64.21 2.145 5 37 37 0(-1) 8(0.067) 9.62 3.78 52.32 1.075 2 38 38 0(-1) 8(0.067) 9.52 3.75 50.14 1.339 2.4 39 39 0(-1) 8(0.067) 9.45 3.69 51.22 1.041 1.6 40 40 0.5(-0.6) 8(0.067) 7.6 3.16 65.22 1.543 1.5 41 41 0.5(-0.6) 8(0.067) 7.54 2.95 63.85 1.496 1.7 42 42 0.5(-0.6) 8(0.067) 7.25 2.96 62.15 1.518 1.3 43 43 1(-0.2) 8(0.067) 6.81 2.75 64.86 1.261 2 44 44 1(-0.2) 8(0.067) 6.52 2.59 62.33 1.295 2.3

111

45 45 1(-0.2) 8(0.067) 6.1 2.65 60.54 1.195 1.7 46 46 1.5(0.2) 8(0.067) 5.01 2.38 64.6 2.139 3.5 47 47 1.5(0.2) 8(0.067) 4.65 2.34 63.29 1.965 3.8 48 48 1.5(0.2) 8(0.067) 4.85 2.15 62.66 2.354 3.2 49 49 2(0.6) 8(0.067) 3.62 1 58.13 2.565 7.1 50 50 2(0.6) 8(0.067) 3.56 1 55.82 2.592 7.2 51 51 2(0.6) 8(0.067) 3.45 1 57.46 2.663 7 52 52 2.5(1) 8(0.067) 3.01 1 54.86 2.507 5.5 53 53 2.5(1) 8(0.067) 2.95 1 52.61 2.658 5 54 54 2.5(1) 8(0.067) 2.78 1 55.83 2.214 6 55 55 0(-1) 12(0.6) 11.48 5.17 48.65 0.475 1.2 56 56 0(-1) 12(0.6) 11.25 5.05 45.18 0.337 1.3 57 57 0(-1) 12(0.6) 10.96 4.95 41.21 0.321 1.1 58 58 0.5(-0.6) 12(0.6) 8.55 4.41 51.99 0.909 1.5 59 59 0.5(-0.6) 12(0.6) 8.21 4.35 52.09 0.958 1.9 60 60 0.5(-0.6) 12(0.6) 8.65 4.25 50.64 0.861 1.1 61 61 1(-0.2) 12(0.6) 8.03 2.5 55.72 1.355 1.2 62 62 1(-0.2) 12(0.6) 7.95 2.35 54.68 1.339 1.3 63 63 1(-0.2) 12(0.6) 7.85 2.25 52.16 1.254 1.1 64 64 1.5(0.2) 12(0.6) 6.13 2.32 63.47 1.936 1.4 65 65 1.5(0.2) 12(0.6) 5.96 2.25 61.95 1.99 1.6 66 66 1.5(0.2) 12(0.6) 6.11 2.06 59.76 1.854 1.2 67 67 2(0.6) 12(0.6) 3.98 2.34 62.75 2.066 6.8 68 68 2(0.6) 12(0.6) 3.58 2.15 60.22 2.054 7 69 69 2(0.6) 12(0.6) 3.75 2.5 59.17 1.965 6 70 70 2.5(1) 12(0.6) 3.25 2.02 58.73 1.549 4.2 71 71 2.5(1) 12(0.6) 3.05 1.99 55.43 1.543 4.4 72 72 2.5(1) 12(0.6) 2.99 1.85 56.33 1.354 4.5 73 73 0(-1) 15(1) 12.52 6.12 42.65 0.325 1 74 74 0(-1) 15(1) 11.96 6.06 39.18 0.187 1 75 75 0(-1) 15(1) 11.24 5.98 35.21 0.171 1 76 76 0.5(-0.6) 15(1) 9.68 4.99 45.99 0.759 1 77 77 0.5(-0.6) 15(1) 9.25 5.21 46.09 0.808 1 78 78 0.5(-0.6) 15(1) 8.96 5.11 44.64 0.711 1 79 79 1(-0.2) 15(1) 8.96 3.25 49.72 1.205 1 80 80 1(-0.2) 15(1) 9.02 3.16 48.68 1.189 1 81 81 1(-0.2) 15(1) 9.62 3.1 46.16 1.104 1 82 82 1.5(0.2) 15(1) 6.99 2.86 57.47 1.786 1 83 83 1.5(0.2) 15(1) 6.26 2.72 55.95 1.84 1 84 84 1.5(0.2) 15(1) 7.06 2.92 53.76 1.704 1 85 85 2(0.6) 15(1) 4.56 2.66 56.75 1.916 4 86 86 2(0.6) 15(1) 5 2.59 54.22 1.904 3 87 87 2(0.6) 15(1) 4.69 2.34 53.17 1.815 4 88 88 2.5(1) 15(1) 4.03 2.35 52.73 1.399 4 89 89 2.5(1) 15(1) 4.27 2.15 49.43 1.393 2 90 90 2.5(1) 15(1) 3.99 2.22 50.33 1.204 5

Table 18. Experimental design for RTC drumstick

Std Run Factor 1 A:Dose(kGy)

Factor 2 B: Storage period (d)

TPC (Log

CFU/g)

YMC (Log

CFU/g) a

value Firmness

(N) Overall

acceptability

1 1 0(-1) 0(-1) 5.613 2.864 -4.5 7.46 8 2 2 0(-1) 0(-1) 5.426 2.694 -4.5 7.23 7.5 3 3 0(-1) 0(-1) 5.501 2.774 -4.5 7.3 7.5 4 4 0.5(-0.6) 0(-1) 2.954 1.589 -4.5 6.88 7 5 5 0.5(-0.6) 0(-1) 3.614 2.109 -4.5 6.81 7.5 6 6 0.5(-0.6) 0(-1) 2.931 1.842 -4.5 6.69 7 7 7 1(-0.2) 0(-1) 2.662 1.265 -4.5 7.38 7 8 8 1(-0.2) 0(-1) 2.601 1.291 -4.5 7.14 7.5 9 9 1(-0.2) 0(-1) 2.706 0.956 -4.5 7.22 7

10 10 1.5(0.2) 0(-1) 2.109 0.782 -4.5 6.62 6 11 11 1.5(0.2) 0(-1) 2.236 0.891 -4.5 6.35 6.5 12 12 1.5(0.2) 0(-1) 2.365 1 -4.5 6.42 6.5 13 13 2(0.6) 0(-1) 0.756 0.295 -4.5 6.32 6 14 14 2(0.6) 0(-1) 0.702 0.383 -4.5 6.25 5.9 15 15 2(0.6) 0(-1) 0.824 0.364 -4.5 6.14 6 16 16 2.5(1) 0(-1) 0.262 0.195 -4.5 5.88 5.5 17 17 2.5(1) 0(-1) 0.278 0.236 -4.5 5.87 5.5 18 18 2.5(1) 0(-1) 0.283 0.264 -4.5 5.88 5.9 19 19 0(-1) 5(-0.33) 6.281 2.953 -3 6.61 5 20 20 0(-1) 5(-0.33) 6.255 2.576 -3 6.84 4.5 21 21 0(-1) 5(-0.33) 6.093 2.994 -3 6.67 5

112

22 22 0.5(-0.6) 5(-0.33) 4.125 2.105 -3 6.54 5.5 23 23 0.5(-0.6) 5(-0.33) 4.198 1.963 -3 6.59 5 24 24 0.5(-0.6) 5(-0.33) 4.396 2.201 -3 6.76 5 25 25 1(-0.2) 5(-0.33) 3.836 2.054 -4.5 7.25 7 26 26 1(-0.2) 5(-0.33) 3.725 2.114 -4.5 6.92 6.5 27 27 1(-0.2) 5(-0.33) 3.694 1.462 -4.5 7.14 6.5 28 28 1.5(0.2) 5(-0.33) 3.274 1.102 -4.5 6.57 5 29 29 1.5(0.2) 5(-0.33) 3.214 0.901 -4.5 6.4 5.5 30 30 1.5(0.2) 5(-0.33) 3.193 0.707 -4.5 6.37 5 31 31 2(0.6) 5(-0.33) 0.892 0.521 -4.5 5.81 4.5 32 32 2(0.6) 5(-0.33) 0.9 0.613 -4.5 6.06 5 33 33 2(0.6) 5(-0.33) 0.91 0.563 -4.5 5.86 4 34 34 2.5(1) 5(-0.33) 0.569 0.451 -4.5 4.79 4 35 35 2.5(1) 5(-0.33) 0.6321 0.396 -4.5 4.77 4 36 36 2.5(1) 5(-0.33) 0.612 0.391 -4.5 4.81 4 37 37 0(-1) 8(0.067) 7.894 3.348 -2.5 5.52 3 38 38 0(-1) 8(0.067) 7.921 2.865 -2.5 5.32 3 39 39 0(-1) 8(0.067) 7.723 3.287 -2.5 5.41 3 40 40 0.5(-0.6) 8(0.067) 6.653 2.371 -2.5 6.64 4 41 41 0.5(-0.6) 8(0.067) 6.98 2.141 -2.5 6.34 4 42 42 0.5(-0.6) 8(0.067) 6.708 2.196 -2.5 6.59 4 43 43 1(-0.2) 8(0.067) 4.115 2.235 -4.5 7.06 6 44 44 1(-0.2) 8(0.067) 4.257 1.856 -4.5 6.87 6 45 45 1(-0.2) 8(0.067) 4.253 2.176 -4.5 7.09 6 46 46 1.5(0.2) 8(0.067) 3.627 1.784 -4.5 5.08 4 47 47 1.5(0.2) 8(0.067) 3.748 1.654 -4.5 4.77 4 48 48 1.5(0.2) 8(0.067) 3.269 1.765 -4.5 4.87 4 49 49 2(0.6) 8(0.067) 2.664 1.851 -4 3.59 3 50 50 2(0.6) 8(0.067) 2.851 1.844 -4 3.15 3 51 51 2(0.6) 8(0.067) 2.317 1.904 -4 3.17 3 52 52 2.5(1) 8(0.067) 1.564 2.589 -4 3.1 2 53 53 2.5(1) 8(0.067) 1.628 2.612 -4 3.11 2 54 54 2.5(1) 8(0.067) 1.596 2.575 -4 3.17 2 55 55 0(-1) 12(0.6) 8.902 3.512 -2 4.05 1 56 56 0(-1) 12(0.6) 8.602 3.201 -2 3.76 1 57 57 0(-1) 12(0.6) 9.214 2.896 -2 3.82 1 58 58 0.5(-0.6) 12(0.6) 8.458 2.965 -2 3.53 2 59 59 0.5(-0.6) 12(0.6) 8.217 2.331 -2 3.46 2 60 60 0.5(-0.6) 12(0.6) 8.394 2.813 -2 3.52 2 61 61 1(-0.2) 12(0.6) 4.189 2.514 -4 6.47 6 62 62 1(-0.2) 12(0.6) 4.324 2.019 -4 6.35 6.5 63 63 1(-0.2) 12(0.6) 5.014 2.369 -4 6.47 6 64 64 1.5(0.2) 12(0.6) 4.791 1.419 -4 3.17 3 65 65 1.5(0.2) 12(0.6) 4.932 1.126 -4 3.14 3 66 66 1.5(0.2) 12(0.6) 5.147 1.154 -4 3.1 3 67 67 2(0.6) 12(0.6) 2.982 2.204 -3.5 2.06 2 68 68 2(0.6) 12(0.6) 3.014 2.132 -3.5 2.03 2 69 69 2(0.6) 12(0.6) 3.141 2.195 -3.5 1.93 2 70 70 2.5(1) 12(0.6) 2.284 2.965 -3.5 1.86 2 71 71 2.5(1) 12(0.6) 2.096 2.925 -3.5 1.92 2 72 72 2.5(1) 12(0.6) 2.165 2.801 -3.5 1.87 2 73 73 0(-1) 15(1) 9.302 3.925 -1.5 1.77 1 74 74 0(-1) 15(1) 9.225 3.881 -1.5 1.81 1 75 75 0(-1) 15(1) 9.314 3.636 -1.5 1.85 1 76 76 0.5(-0.6) 15(1) 8.958 3.065 -1.5 2.87 1 77 77 0.5(-0.6) 15(1) 8.77 3.231 -1.5 2.74 1 78 78 0.5(-0.6) 15(1) 8.914 3.113 -1.5 2.77 1 79 79 1(-0.2) 15(1) 6.489 2.835 -3.5 6.32 1 80 80 1(-0.2) 15(1) 6.546 2.908 -3.5 6.26 1 81 81 1(-0.2) 15(1) 6.714 2.891 -3.5 6.21 1 82 82 1.5(0.2) 15(1) 5.791 2.619 -3 3.1 1 83 83 1.5(0.2) 15(1) 5.632 2.351 -3 2.94 1 84 84 1.5(0.2) 15(1) 5.947 2.524 -3 2.89 1 85 85 2(0.6) 15(1) 3.812 2.913 -3 1.79 1 86 86 2(0.6) 15(1) 3.951 2.826 -3 1.84 1 87 87 2(0.6) 15(1) 3.824 2.915 -3 1.91 1 88 88 2.5(1) 15(1) 3.834 3.25 -3.1 1.7 1 89 89 2.5(1) 15(1) 3.287 3.225 -3.1 1.67 1 90 90 2.5(1) 15(1) 3.294 3.281 -3.1 1.64 1

113

Table 19. Experimental design for RTC pumpkin

Std Run

Factor 1

A:Dose

(kGy)

Factor 2 B:

Storage

duration (d)

TPC

(Log

CFU/g)

Y & MC

(Log

CFU/g)

Lvalue Firmness

(N)

Overall

acceptability

1 1 0(-1) 0(-1) 5.03 2.69 75.25 1.719 8 2 2 0(-1) 0(-1) 5.52 3.12 74.96 1.655 8.5 3 3 0(-1) 0(-1) 4.97 2.25 75.24 1.769 8.5 4 4 0.5(-0.6) 0(-1) 3.01 1.74 72.38 1.404 8.5 5 5 0.5(-0.6) 0(-1) 3.25 1.77 72.73 1.541 8 6 6 0.5(-0.6) 0(-1) 3.1 1.55 73.06 1.455 8 7 7 1(-0.2) 0(-1) 2.51 1.72 73.91 1.23 7.5 8 8 1(-0.2) 0(-1) 2.17 1.96 74.98 1.121 7 9 9 1(-0.2) 0(-1) 2.97 1.45 73.28 1.23 7.5

10 10 1.5(0.2) 0(-1) 2.21 1.41 75.25 0.907 6.5 11 11 1.5(0.2) 0(-1) 1.54 1.42 74.82 1.064 6.2 12 12 1.5(0.2) 0(-1) 1.82 1.28 73.86 1.025 6 13 13 2(0.6) 0(-1) 1.09 1.03 75.88 0.842 6.5 14 14 2(0.6) 0(-1) 1.52 1.01 75.03 0.768 6 15 15 2(0.6) 0(-1) 1.12 0.92 74.96 0.754 6 16 16 2.5(1) 0(-1) 0.64 0.39 75.26 0.678 5.2 17 17 2.5(1) 0(-1) 0.42 0.34 75.22 0.634 5.5 18 18 2.5(1) 0(-1) 0.63 0.52 74.021 0.724 5 19 19 0(-1) 5(-0.33) 5.35 4.05 79.23 1.766 7.5 20 20 0(-1) 5(-0.33) 4.95 4.59 78.33 1.806 7.2 21 21 0(-1) 5(-0.33) 5.59 3.56 77.8 1.769 7 22 22 0.5(-0.6) 5(-0.33) 3.25 2.62 78.66 1.216 6 23 23 0.5(-0.6) 5(-0.33) 3 2.25 79.61 0.995 6.2 24 24 0.5(-0.6) 5(-0.33) 3.32 3.26 78.98 1.332 6 25 25 1(-0.2) 5(-0.33) 2.78 2.68 80.52 1.125 7 26 26 1(-0.2) 5(-0.33) 2.25 2.35 80.72 1.245 7.5 27 27 1(-0.2) 5(-0.33) 2.99 2.85 81.42 1.235 7 28 28 1.5(0.2) 5(-0.33) 1.83 2.02 82.39 1.038 6.2 29 29 1.5(0.2) 5(-0.33) 2.13 1.88 75.35 0.9321 6 30 30 1.5(0.2) 5(-0.33) 2 0.94 77.93 0.802 6.2 31 31 2(0.6) 5(-0.33) 2.02 1.18 80.97 0.822 5 32 32 2(0.6) 5(-0.33) 1.63 0.96 70.82 0.967 5.2 33 33 2(0.6) 5(-0.33) 2.02 1.96 84.15 0.863 5 34 34 2.5(1) 5(-0.33) 0.93 0.86 72.33 0.654 5.5 35 35 2.5(1) 5(-0.33) 1.91 0.93 77.88 0.797 5 36 36 2.5(1) 5(-0.33) 1.13 1 79.38 0.779 5 37 37 0(-1) 8(0.067) 6.57 4.53 85.87 1.8104 1 38 38 0(-1) 8(0.067) 6.25 4.22 85.31 1.782 1.5 39 39 0(-1) 8(0.067) 7.01 4 86.13 1.955 1 40 40 0.5(-0.6) 8(0.067) 4.07 3.07 86.53 1.425 4 41 41 0.5(-0.6) 8(0.067) 4.56 3.32 84.77 1.04 3.5 42 42 0.5(-0.6) 8(0.067) 3.54 3.74 86.52 1.338 4 43 43 1(-0.2) 8(0.067) 3.63 2.76 84.66 1.128 7 44 44 1(-0.2) 8(0.067) 3.1 2.71 85.45 1.229 6.5 45 45 1(-0.2) 8(0.067) 3.66 2.77 84.57 1.312 7 46 46 1.5(0.2) 8(0.067) 3.06 2.49 81.4 0.729 5 47 47 1.5(0.2) 8(0.067) 2.93 2.82 78.691 1.034 5.2 48 48 1.5(0.2) 8(0.067) 2.68 2.23 89.653 0.934 5 49 49 2(0.6) 8(0.067) 2.37 3 85.965 0.836 4 50 50 2(0.6) 8(0.067) 2.13 1.21 85.319 0.921 3.5 51 51 2(0.6) 8(0.067) 1.96 1.93 76.342 0.768 4 52 52 2.5(1) 8(0.067) 2.15 1.51 74.923 0.759 4 53 53 2.5(1) 8(0.067) 2.01 1.43 79.642 0.734 3 54 54 2.5(1) 8(0.067) 1.83 1.52 85.819 0.603 4 55 55 0(-1) 12(0.6) 6.8 4.61 89.25 1.981 1 56 56 0(-1) 12(0.6) 7.31 4.22 88.39 1.89 1 57 57 0(-1) 12(0.6) 7.01 5.01 95.48 1.947 1 58 58 0.5(-0.6) 12(0.6) 6.51 3.16 89.54 1.245 3 59 59 0.5(-0.6) 12(0.6) 6.96 3.95 83.57 1.321 2.5 60 60 0.5(-0.6) 12(0.6) 6.21 2.96 84.21 1.129 3 61 61 1(-0.2) 12(0.6) 3.93 3.08 85.98 1.108 7 62 62 1(-0.2) 12(0.6) 4.13 3.37 86.21 1.02 6.5 63 63 1(-0.2) 12(0.6) 3.85 3 86.94 0.97 7 64 64 1.5(0.2) 12(0.6) 3.01 2.76 88.642 0.967 4 65 65 1.5(0.2) 12(0.6) 3.48 2.9 87.06 0.797 3 66 66 1.5(0.2) 12(0.6) 2.97 2.96 83.921 0.803 4

114

67 67 2(0.6) 12(0.6) 2.66 2.39 87.406 0.778 3 68 68 2(0.6) 12(0.6) 2.72 2.42 79.693 0.876 2.5 69 69 2(0.6) 12(0.6) 2.53 2.26 89.335 0.783 3 70 70 2.5(1) 12(0.6) 2.2 1.5 82.721 0.669 3 71 71 2.5(1) 12(0.6) 2.19 1.48 78.745 0.686 2.5 72 72 2.5(1) 12(0.6) 2.31 1.63 89.241 0.608 3 73 73 0(-1) 15(1) 9.85 6.4 96.21 2.1 1 74 74 0(-1) 15(1) 9.53 5.99 93.25 2.321 1 75 75 0(-1) 15(1) 9.07 6.21 90.28 2.206 1 76 76 0.5(-0.6) 15(1) 8.56 4.96 92.54 1.1235 1 77 77 0.5(-0.6) 15(1) 7.97 5.21 84.57 1.021 1 78 78 0.5(-0.6) 15(1) 7.87 5.17 85.21 0.987 1 79 79 1(-0.2) 15(1) 4.87 4.12 86.98 0.963 2 80 80 1(-0.2) 15(1) 5.96 3.99 87.21 0.859 2 81 81 1(-0.2) 15(1) 5.57 4.37 87.94 0.821 2 82 82 1.5(0.2) 15(1) 3.97 3.27 89.642 0.765 3 83 83 1.5(0.2) 15(1) 3.67 4.33 88.06 0.652 2.5 84 84 1.5(0.2) 15(1) 4.13 4.22 84.921 0.506 3 85 85 2(0.6) 15(1) 3.13 3.96 88.406 0.521 2 86 86 2(0.6) 15(1) 2.97 3.36 80.693 0.536 1.5 87 87 2(0.6) 15(1) 3.66 3.02 90.335 0.468 2 88 88 2.5(1) 15(1) 2.32 2.57 83.721 0.521 2 89 89 2.5(1) 15(1) 2.85 2.21 79.745 0.511 1 90 90 2.5(1) 15(1) 1.97 2.2 90.241 0.421 2

Table 20. Significance statistics, p values and signal to noise ratio (S/N) of predicted

models for ash gourd

Model Lack of fit S/N

Response Sum of

squares

df p-value Sum of

squares

df p-value

TPC 742.38 7 0.0001 14.62 20 0.2501 65.713

Y&MC 155.62 9 0.0001 9.23 20 0.0621 49.464

Color (L) 4988.54 7 0.0001 440.88 22 0.07801 39.457

Firmness 55.79 8 0.0001 5.35 21 0.0001 40.374

Overall

acceptability

469.62 9 0.0001 58.09 20 0.3243 23.791

115


models for drumstick


Response Sum of

squares

df p-value Sum of

squares

df p-value

TPC 505.63 5 0.0001 13.89 24 0.3558 72.85

Y&MC 270.24 6 0.0001 18.18 23 0.0515 45.34

Color (a) 72.65 7 0.0001 12.66 22 0.0831 29.03

Firmness 524.69 6 0.0001 53.85 23 0.073 34.22

Overall

acceptability

675.48 8 0.0001 32.99 21 0.0511 42.44


models for pumpkin


Response Sum of

squares

df p-value Sum of

squares

df p-value

TPC 135.7176 8 0.0001 2.826047 15 0.1647 50.440

Y&MC 80.52517 6 0.0001 2.212739 17 0.4606 36.465

Color (L) 1652.848 3 0.001 97.26419 20 0.9714 22.343

Firmness 7.21269 6 0.0001 0.229633 17 0.1097 34.694

Overall

acceptability

222.4349 7 0.0001 47.0651216 16 27.306

116

Table 23. Coefficients of the fitted polynomial representing the relationship between the

response and the process variable and R2 values for RTC ash gourd

Coefficients TPC

(log cfu/g)

Y & MC

(log cfu/g)

Color (L) Firmness (N) Overall

acceptability

Intercept 5.43a 1.97

a 63.30

a 2.21

a 3.75

a

A-Dose -3.56a -1.13

a 1.12

a 0.66

a 4.09

a

B-Storage time 2.08a 1.20

a -7.04

a -1.30

a -3.10

a

AB -1.29a -0.67

a 4.63

a 0.71

a 1.20

a

A2

0.38a 0.39

a -5.48

a -0.38

a 0.055

a

B2 -0.51

a 0.22

a -2.64

a 0.00079 0.70

a

A2B 0.10

a 0.58

a -4.15

a -0.35

a 1.33

a

AB2 0.83

a -0.18

a 1.91

a -0.35

a -2.34

a

A3

0.099 a 0.019

a 0.01 -0.35

a -1.90

a

B3 0.43

a -0.19

a -0.01 0.64

a -0.53

a

R2 0.97 0.93 0.89 0.90 0.87

a Significant terms at p ≤ 0.05


response and the process variable and R2 values for RTC drumstick

Coefficients TPC

(log cfu/g)

Y & MC

(log cfu/g)

Color (a) Firmness (N) Overall

acceptability

Intercept -0.39a -0.51

a -4.07

a 1.46

a 0.91

a

A-Dose -0.68a -0.56

a -1.12

a -1.61

a -1.45

a

B-Storage time 0.57a 0.57

a 0.59

a -1.27

a -0.49

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AB 0.018 1.15a -0.38

a -0.056 2.97

a

A2

-0.100 0.62a 0.72

a -1.58

a -1.90

a

B2 0.12 0.019

a 0.18

a -0.74

a 1.49

a

A2B 2.49

a 1.83

a 0.45

a -2.96

a -2.19

a

AB2 -1.72

a -1.25

a 0.48

a -0.96

a -2.13

a

A3

-1.24a -0.34 0.27 0.64

a 2.66

a

B3 0.22 0.26 0.14 0.073

a -1.24

a

R2 0.92 0.86 0.86 0.79 0.89



response and the process variable and R2 values for RTC pumpkin

Coefficients Logit(TPC) Y&MC

(log cfu/g)

Color (L) Firmness

(N)

Overall acceptability

(Hedonic)

Intercept 0.037187a 3.15638

a 82.04992

a 0.275679

a 5.737103

a

A-Dose -0.87613a -0.73809

a -0.80918

a -0.2271

a 1.074074

a

B-Storage time 0.984792a 0.857211

a 8.792812

a -0.00235 -3.42333

a

AB -0.43095a -0.04779 -0.8359

a -0.04877

a 1.412679

a

A2

0.313907a 0.223595

a 0.484688 0.051385

a -0.8006

a

B2 0.578462

a -0.43183

a -1.39156 0.018442 0.3375

A2B 0.120244

a -0.00117 0.048437 0.014139

a -0.30804

a

AB2 -0.29278

a 0.100796

a 0.352516 -0.02633

a -0.225

A3

-0.04786a -0.04214

a -0.12251 -0.008

a 0.113426

a

B3 -0.10923 -0.02724 -1.78247 -0.02758 1.3125

a

R2 0.937315 0.905814 0.730179 0.918311 0.825361


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3.1.1.1. Microbial quality

Microbial contamination can be a major source of spoilage of fresh-cut produce.

Microbial spoilage has been used by quality assurance departments in the fresh-cut

industry as an objective indicator of quality failure of fresh-cut vegetable commodities

[147]. Contamination sources of fresh-cut produce include raw materials and contact with

processing equipment. The micro-organisms that exist on the surfaces of raw, whole

produce appear to be the major source of microbial contamination and consequent

spoilage of fresh-cut fruits and vegetables.

Fresh-cut vegetables have high levels of microorganisms and several outbreaks of food-

borne illnesses have been found to be associated with these products (Fan et al., 2008

from intro). In general, total bacterial counts of fresh-cut products just after processing

were reported to range from 3 to 6 log CFU g-1

[13]. There are no standard prescribed

limits for acceptable microbial load in case of fresh fruits and vegetables. The upper limit

for total microbial load of fresh-cut vegetables during the entire intended storage duration

as set by European Standards is < 7 log CFU g-1 [148]. However, according to guidelines

of the Centre for Food Safety, Hong Kong, the total microbial load of < 5 log CFU g-1

has been considered satisfactory for fresh-cut mixed salads [149]. Therefore, in the

present study, a total microbial load of 5 log CFU g-1 was taken as the upper limit for

microbial acceptability of RTC vegetables.

A significant (p ≤ 0.05) increase in mesophilic bacterial (TPC) and yeast and mold counts

(Y&MC) during storage was observed in non-irradiated control samples of all the three

vegetables. Gamma irradiation significantly reduced microbial load and it decreased

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linearly with radiation dose. The effect of radiation treatment and storage duration on

TPC and Y&MC in ash gourd, drumstick and pumpkin is depicted in Figures 12, 13 and

14, respectively.

Figure 12. Variation of (a) Total plate counts (media: plate count agar, 37 °C, 24 h) and

(b) yeast and mold counts (media: potato dextrose agar, 37 °C, 48 h) with radiation dose

(0 – 2.5 kGy) and storage time (0 – 15 d) in RTC ash gourd.



(0 – 2.5 kGy) and storage time (0 – 15 d) in RTC drumstick.

120



(0 – 2.5 kGy) and storage time (0 – 28 d) in RTC pumpkin.

Ash gourd: Control samples of RTC ash gourd on zero day of storage had TPC and

Y&MC load of 3.86 and 2.09 log CFU/g, respectively. The mesophilic counts reduced by

2-3 log cycles compared to the control immediately after radiation treatment depending

on the dose delivered (Figure 12 a). In control and samples irradiated at 0.5 and 1 kGy,

counts reached higher than 7 log CFU/g, on day eight, beyond the acceptable limit

prescribed (5 log CFU/g) for fresh cut vegetables [148, 149]. In samples exposed to a

radiation dose of 1.5 kGy the mesophilic counts showed values greater than 5 log CFUg-1

on day 12 of storage period, reducing the microbial quality of the product. Samples

irradiated at 2 kGy or above exhibited no microbial spoilage until 12 d of storage. At the

end of storage period (15 d), the counts exceeded the acceptable limit in control and

samples irradiated below 2 kGy. However, a radiation dose of 2 kGy and above was

suitable to keep the mesophillic counts well below the acceptable limit.

121

The response of yeast and mold counts for different radiation doses is represented in

Figure 12 b. At 2 and 2.5 kGy, a reduction in counts by 2 log cycles was observed after

irradiation on day zero. The low counts were maintained during storage in these samples

as compared to the control and those irradiated at lower doses (0.5–1.5 kGy). Thus

radiation treatment at 2 kGy and above was highly effective in reducing total mesophilic,

and yeast & mold counts in RTC ash gourd.

Drumstick: The effect of radiation processing and storage duration on TPC and Y&MC

load in RTC drumstick is depicted in Figure 13. On day zero, TPC and Y&MC load of

control samples were 5.61 and 2.86 log CFU/g, respectively. Significant (p < 0.05) dose

dependent reduction in mesophilic bacterial counts was observed in radiation processed

samples. The mesophilic counts reduced by 3-5 log cycles immediately after radiation

treatment compared to the control. A 3-log reduction was observed in mesophilic counts

at a radiation dose of 1 kGy, while Y&MC decreased by 2 log cycles at this dose. During

storage, a significant (p < 0.05) increase in TPC and Y&MC was observed in control as

well as irradiated samples. However, radiation processed samples had a significantly

lower counts as compared to control at all the storage days studied. At the end of storage

period (15 d), the TPC exceeded the acceptable limit in control (> 9 log CFU/g) and

samples irradiated at 0.5 kGy. However, a radiation dose of 1 kGy and above was suitable

to keep the mesophillic counts well below the acceptable limit.

Pumpkin: The mesophilic bacterial and Y&MC counts were 5.12 and 2.7 log CFU/g

respectively in control pumpkin on day zero. The effect of radiation treatment and storage

duration on TPC and Y&MC is depicted in Figure 14. Radiation processing resulted in a

122

significant (p < 0.05) dose-dependent reduction in the microbial counts. A 4-log reduction

was observed in mesophilic bacterial counts at a radiation dose of 2 kGy, while Y&MC

decreased by 2 log cycles at equivalent radiation dose. During storage, a significant

increase in both TPC and Y&MC was observed (p < 0.05) in control as well as radiation-

treated samples. However, radiation processed samples at all doses had significantly (p <

0.05) lower microbial counts as compared to control throughout the storage period. At the

end of storage period (28 d) TPC was > 9 log CFU/g for control samples while TPC for

radiation-treated (2 kGy) samples was < 4 log CFU/g.

When ionizing radiation strikes bacteria and other microbes, its high energy breaks

chemical bonds in molecules that are vital for cell growth and integrity. As a result, the

microbes die, or no longer multiply to cause spoilage [150]. Radiation induced reduction

of microbial load by 5-log cycle in french beans at a dose of 2 kGy [122], and a 2 log

cycle reduction in radiation-treated celery (1.5 kGy) was observed [151]. Reductions of

approximately 5.25 and 4.14 log CFU/g of a five-strain cocktail of L. monocytogenes

were reported for cabbage and tomato, respectively, at a radiation dose of 1 kGy [88,152]

reported that the initial bacterial load in control carrot samples (Daucus carota) was 6.3 ×

102 CFU/g in control samples which increased to 6.5 × 10

5 CFU/g after 14 days of

storage. A dose of 1 kGy reduced the bioload down to 12.0 CFU/g, with few colonies

after 14 d storage. The samples receiving 2 kGy or higher doses were found to be

completely free of bacteria during 14 days of refrigerated storage. Thus, the results

obtained in the present study are in accordance with already published literature data.

123

It is proposed that elimination of spoilage bacteria can bring about enhanced growth of

some pathogenic bacteria. Reports however document 4–5 log cycle reduction of L.

monocytogenes in radiation processed sprouts [153] and watercress [154]. Thus

pathogenic microbes were not monitored in the present study.

3.1.1.2. Color

The three Commission Internationale de l’Eclairage (CIE) co-ordinates “L*”, “a*” and

“b*” values were measured for all the three vegetables. “L*” value represents luminosity

of the sample and varies from 0 (black) to 100 (white). Increase in luminosity can be

correlated with the development of whiteness in the samples, while a decrease of

luminosity can be an indication of browning appearance. In case of ash gourd and

pumpkin, significant (p ≤ 0.05) effect of radiation treatment and storage was observed in

“L*” values, however, no significant (p ≤ 0.05) change in “a*” and “b*” values was

noticed. On the other hand, for processed drumstick samples, significant changes were

noticed in “a*” values (+ red, - green) while “L*” and “b*” values were not affected.

Therefore “L*” values for ash gourd and pumpkin, and “a*” values for RTC drumstick

were analyzed with respect to radiation treatment and storage. Figure 15 depicts the effect

of radiation treatment and storage on the color attributes of ash gourd, drumstick and

pumpkin.

Ash gourd: The effect of radiation processing and storage duration on L* values is

depicted in figure 15 (a). L* values continuously decreased with storage time in the

control samples. Beyond 5 d, the control samples exhibited appreciable lowering in L*

values and visual browning that increased during further storage. L* values also

124

decreased for samples irradiated at lower doses (0.5–1.5 kGy) after 8 d. It is interesting to

note that the luminosity (L*) of the cut ash gourd irradiated at 2 and 2.5 kGy remained

unchanged during storage and the visual quality was acceptable at the end of storage

period (12 d).

Figure 15. Effect of radiation treatment and storage on (a) L values of RTC ash gourd; (b)

a values of RTC drumstick; (c) L values of RTC pumpkin.

Radiation induced inhibition in surface browning has been documented by [155] in fresh-

cut vegetables. Decrease in surface browning of lettuce with increasing radiation dose

was also reported by Fan et al [94, 156]. A negative correlation between browning

intensity and lightness (L* value) has been documented [157]. Polyphenoloxidase (PPO)

is a key enzyme responsible for browning in fruits and vegetables, which catalyzes

125

oxidation of phenolic compounds, resulting in formation of colored melanins. Radiation

treatment might alter the reactions catalyzed by these enzymes, thus limiting the

development of brown pigments.

Drumstick: On day zero, no significant (p ≤ 0.05) effect of radiation processing was

observed on the “a*” values of cut drumstick samples. A positive value of ‘a*’ indicates

increased perception of redness, while negative values indicate greenness. During

storage, “a*” (-ive) values decreased with increasing storage period in control samples

(Figure 15 b), indicating a reduction in the greenness. Reduction of greenness might be

due to degradation of pigments during storage. Higher “a*” values (-ive) were obtained

for radiation treated samples as compared to control samples during the entire storage

duration indicating better retention of greenness throughout the intended storage period.

Pumpkin: The effect of radiation treatments and storage on L values of RTC pumpkin is

depicted in Figure 15 (c). Radiation processing had no significant (p < 0.05) effect on L*

values. A significant (p < 0.05) increase in L* values was however observed for both

control and radiation-treated samples with increasing storage duration (0 to 28 d).

Moreover, during storage the change in L* values for radiation processed samples was

significantly lower as compared to control. The L* value on day zero was 75.0 ± 3.23

which increased to 96.0 ± 5.48 and 83.0 ± 4.16 on day 28 for control and irradiated (2

kGy) samples, respectively. An increase in L* values signifies a decreased colour

intensity of control pumpkin samples. A similar trend was reported earlier in Citrus

clementina [158] and french beans [122].

126

In general, during extended storage, fading or decreased color intensity has been reported

in various fresh-cut products. Degradation of pigments during storage resulting in fading

of the color has been suggested as the probable reason for this observation [122].

Reduced change in color attribute (L*, a* or b* value) of radiation treated samples during

storage as compared to control might be due to lower rate of respiration and physiological

changes, thus resulting in better retention of pigments and higher color intensity during

storage.

3.1.1.3. Texture

Minimally processed vegetables that maintain firm, crunchy texture are highly desirable

because consumers associate these textures with freshness and wholesomeness [159].

During mechanical operations, cut surfaces are damaged, releasing enzymes which spread

through the tissue and come into contact with their substrates. The softening of fresh-cut

fruit is mainly due to the enzymatic degradation of the cell wall, which is mainly

composed of cellulose, hemicellulose and pectins. The loss of desirable texture in fresh-

cut products is a major problem. It was therefore of interest to study the effect of radiation

processing and storage on firmness of the RTC vegetables.

Ash gourd: A significant (p ≤ 0.05) effect of storage time and radiation processing on the

firmness of samples was observed (Figure 16 a). Irradiation resulted in decreased

firmness (p ≤ 0.05) at all the doses studied on day zero. During storage, continuous

decrease in firmness was observed in control and samples irradiated at lower doses (0.5–

1.5 kGy). However, in samples irradiated at 2 and 2.5 kGy, the firmness quality was not

significantly (p ≤ 0.05) affected during storage.

127

Figure 16. Effect of radiation treatment and storage on firmness of (a) Ash gourd; (b)

Drumstick; (c) Pumpkin.

Drumstick: Effect of radiation processing and storage duration on the firmness of cut

drumstick is depicted in Figure 16 (b). A dose dependent decrease in firmness was

observed in radiation processed samples on day zero. In non-irradiated and samples

irradiated at 0.5 kGy, firmness decreased continuously during the storage. At 1 kGy, the

firmness was not significantly affected as a result of storage. At doses > 1.0 kGy,

significant softening was observed immediately after irradiation which increased further

during storage.

Pumpkin: A radiation dose-dependent reduction in firmness of RTC pumpkin cubes was

observed on day zero with a 50 % reduction at 2.0 kGy (Figure 16 c). Interestingly, an

128

increase in firmness of control samples was observed during storage (1.5 ± 0.2 N at 0 d to

2.1 ± 0.2 N at 28 d). However, no significant increase in firmness during storage was

noticed in radiation-treated samples. This could possibly be due to the lower respiration

rate in radiation treated samples resulting in decreased water loss and subsequently better

maintenance of texture. The firmness of the developed radiation-treated product was

therefore maintained during the entire intended storage while control samples exhibited

increased firmness after seven days of storage.

Radiation processing is known to induce softening immediately after treatment. Such

observations have been previously attributed to radiation-induced hydrolysis of pectin and

other cell wall components such as cellulose and hemicellulose [160]. No significant

changes in texture during storage have been reported in various radiation-treated fresh-cut

produce such as celery, green leaf lettuce, iceberg lettuce, parsley and red leaf lettuce by

Fan & Sokorai, 2008 [12]. The better retention of texture of radiation processed samples

during storage could possibly be due to lower rate of respiration in plant produce which

results in decreased water loss and subsequently better firmness [161].

On the other hand, increased firmness in fresh-cut tissues during storage has also been

reported earlier in some cases such as ready-to-use carrots, where a significant increase in

firmness was observed during storage [162]. Lignification was cited as the probable

reason by these researchers. Due to removal of outer epidermal layer to form peeled

ready-to-use product, physiological changes occur in the vegetable to initiate the

production of a new protective layer of lignin, giving a stiff aspect to the fresh-cut

vegetable and requiring greater force to break the plant tissue. Also water loss in fresh-cut

129

produce is known to result in drying of the outer layers and hence requiring more force to

pierce into the tissue [162].

3.1.1.4. Sensory acceptability

Consumer integrates all sensory inputs - appearance, texture, off-flavours and odours -

into a final judgement of the acceptability of that fruit or vegetable. In order to get an

appropriate implementation of a treatment for preservation or improvement in shelf life,

sensory evaluations need to be carried out to ensure that the perceived quality of a

determined product is not negatively affected. The sensory analysis was therefore

conducted to study the effect of radiation treatment and storage on consumer

acceptability.

Figure 17. Effect of radiation treatment and storage on the overall sensory acceptability of

(a) Ash gourd; (b) Drumstick; (c) Pumpkin.

130

Ash gourd: The results of the hedonic test for the overall acceptability of the product are

depicted in Figure 17 a. No significant difference (p ≤ 0.05) in hedonic scores of control

and irradiated samples up to a dose of 1.5 kGy was noted after irradiation on day zero.

Samples irradiated at 2 kGy and above exhibited softening in texture, resulting in lower

scores, however it was statistically insignificant. The sensory quality of control samples

was found to deteriorate within 5 d of storage. Between radiation doses 0.5–1.5 kGy, the

acceptability of the samples decreased with storage. However, at 2 and 2.5 kGy, it

remained unchanged during entire storage period. Interestingly, the acceptability of

samples at 2 kGy was higher than that of 2.5 kGy at all stages of storage. This could be

due to better retention of texture in samples irradiated at 2 kGy as compared to that of 2.5

kGy. These samples (2 kGy) also scored better in aroma and taste attributes making them

more acceptable compared to the other samples. Hedonic analysis thus indicated that

radiation processed samples at a dose of 2 kGy had the highest scores throughout the

storage period.

Drumstick: Figure 17 b shows the results of hedonic testing for overall acceptability of

control and radiation processed drumstick samples. The overall acceptability of the

samples was unaffected up to a radiation dose of 1.0 kGy, while it decreased at doses >

1.0 kGy on day zero. The hedonic scores obtained for control and samples subjected to

radiation doses of 0.5 and 1.0 kGy were 8.0 ± 0.3, 7.5 ± 0.5 and 7.0 ± 0.5 respectively,

while that for irradiated at 1.5, 2.0 and 2.5 kGy lied between 5 and 6. This might be

attributed to decreased firmness of the samples at higher doses. The acceptability of

control and samples irradiated at 0.5 kGy decreased drastically from day 5 onwards. This

131

may be attributed to reduced aroma and texture scores obtained. The samples irradiated

at 1 kGy exhibited excellent sensory quality during storage period up to 12 d with a

sensory score of 7 ± 0.5 for overall acceptability; however, the sensory score decreased to

< 4 on day 15.

Pumpkin: The hedonic scores plotted against radiation doses and storage duration is

shown in Figure 17 c. On day zero, the effect of radiation dose up to 1 kGy on the

hedonic scores was insignificant (p < 0.05). The values obtained were, respectively, 8.0 ±

0.7, 7.5 ± 0.5 and 7.0 ± 0.25 for control and samples subjected to radiation doses of 0.5

and 1.0 kGy. Sensory scores for samples irradiated at higher doses (beyond 1 kGy) were

significantly lower as compared to control on day zero. Scores for these samples were

between 4.5 and 5.5. This might be attributed to decreased firmness perceived by sensory

panel at these doses. Decrease in firmness is also evident from the instrumental data for

firmness (Figure 16 c). The acceptability of control and irradiated samples (0.5 kGy)

decreased drastically from day 14 onwards. This could be explained on the basis of

reduced aroma notes perceived by sensory panel and harder texture of the samples

towards the end of storage resulting in lower scores. The samples irradiated at 1 kGy

exhibited excellent sensory quality during storage period up to 21 d with a sensory score

of 6 ± 0.5 for overall acceptability; however, the sensory score decreased to < 3 on day

28. Significantly higher aroma scores for radiation processed carrots, cilantro, green

onions, parsley and red lettuce after 14 days of storage have been reported earlier [12].

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3.1.1.5. Optimization and verification of results

The processing factors were optimized for achieving maximum shelf-life of the RTC

products with acceptable sensory and microbial quality. Apart from this, the optimized

levels of the variables and the importance of the responses were also taken into account.

Criteria set for optimization and solutions obtained for the individual vegetable product

are depicted in Table 26. The criteria decided aimed at maximizing shelf life of product

with mesophillic load of < 5 log CFU/g, minimum change in color & texture and an

overall acceptability score of greater than 5.

Table 26. Criteria for various factors and responses for process optimization and

corresponding optimized solutions obtained

Name Criteria 1 Criteria 2 Solution A Solution B

Ash gourd

A:Dose (kGy) minimize minimize 2.34 1.95

B:Storage time (d) maximize (8-15) maximize (5-12) 14 12

TPC (Log CFU/g) minimize (<5) is in range (<6) 3.95a; (3.76 ± 0.58)

b 3.92a; (3.86 ± 0.41)

b

Y&M (Log CFU/g) minimize minimize 2.35a; (2.28 ± 0.21)

b 1.60a; (1.52 ± 0.22)

b

Color (L) In range (39.0-

68.0)

In range (45- 68) 53.15a; (57.56 ±

2.54)b

62.09a; (61.29 ± 4.67)

b

Firmness (N) maximize(1.6-3.5) in range (1.0-3.5) 1.75a; (1.84 ± 0.50)

b 2.32a; (2.15 ± 0.35)

b

Overall

acceptability

(Hedonic)

In range (5-7) ≥ 5 5.52a; (5.65 ± 0.25)

b 5.38a; (5.21 ± 0.25)

b

Drumstick



TPC (Log CFU/g) Minimize (<5) In range (<6) 3.33a; (3.52 ± 0.52)b 4.99a;(5.10 ± 0.45)

b

Y&M (Log CFU

/g) minimize minimize 1.33a; (1.62 ± 0.42)

b 2.28a; (2.12 ± 0.54)

b

Color (a) In range (-4.5- -1.5) In range (-4.5- -

1.5) -4.32a; (-4.45 ± 0.98)

b -3.25a; (-3.14 ± 0.41)

b

Firmness (N) In range (4.41-7.45) maximize(0.6-1.0) 4.26a; (4.59 ± 1.55)b

3.65a; (3.88 ± 1.25)b

Overall

acceptability

(Hedonic)

≥ 5 ≥ 5 5.36a; (5.3 ± 0.4)b

6.00a; (5.5 ± 0.6)b

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Pumpkin



TPC (Log CFU/g) minimize (< 5) is in range (< 6) 3.79a;(4.12 ± 0.45)

b 4.35

a; (4.92 ± 0.52)

b

Y&M (Log CFU

/g)

minimize minimize 2.91a; (3.12 ± 0.54)

b 3.02

a; (3.62 ± 0.42)

b

Color (L) minimize minimize 87.11a; (82.14 ±

5.41)b

87.65a; (90.14 ± 4.98)

b

Firmness (N) is in range

(0.6-1.0)

Maximize

(>0.8 <1.7)

0.96a; (0.89 ± 0.12)

b 1.04

a; (0.99 ± 0.21)

b

Overall

acceptability

(Hedonic)

In range (5-8) ≥ 5 5.26a; (5.5 ± 0.6)

b 5.18

a; (5.3 ± 0.4)

b

aPredicted values for solutions.

bActual values.

Two solutions were obtained for each vegetable depending on the criteria decided.

Solution A and B were obtained for criteria 1 and 2 respectively. For ash gourd Solution

A suggested a radiation dose of 2.34 kGy for a shelf life of 14 d while a shelf life of 12 d

was suggested at 2 kGy dose in solution B. For RTC drumstick, solutions A and B

suggested a radiation dose of 1.5 and 1.0 kGy for a shelf life of 7 and 12 d respectively. In

case of pumpkin, Solution A and B suggested a radiation dose of 1.2 and 1 kGy,

respectively, with a storage period of 21 d.

Models generated were validated using both the solutions obtained for each vegetable

separately. For the validation experiments, fresh samples of ash gourd, drumstick and

pumpkin were cut, packaged and irradiated at doses suggested in solutions obtained (2.3

and 2.0 kGy for ash gourd; 1.5 and 1.0 kGy for drumstick; 1.2 and 1.0 kGy for pumpkin).

They were subsequently analyzed for microbial quality (TPC and Y&MC), color, texture

and sensory acceptability during the intended storage period as obtained in solutions A

and B. A good agreement of actual and predicted values indicated the suitability of the

models (Table 26).

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Both the solutions obtained for the three vegetables demonstrated a microbial load < 5 log

CFU/g and acceptable sensory quality (overall acceptability above 5). The solutions

corresponding to the lower radiation doses with acceptable microbial and sensory quality

were selected for each vegetable. Thus an optimum dose of 2 kGy and shelf life of 12 d

for ash gourd, and a dose of 1 kGy with a shelf life of 12 and 21 d for drumstick and

pumpkin respectively, was chosen. The products processed at these radiation doses were

further analyzed for the nutritional quality in terms of radical scavenging activity, total

phenolic and flavonoid content, vitamin C content, head space gaseous composition and

quantitative descriptive sensory analysis (QDA). Bioactive constituents were also

analyzed for the developed products at the optimized dose.

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3.2. Characterization of the developed RTC products

Figure 18 presents the control and radiation processed products of RTC ash gourd,

drumstick and pumpkin.

Figure 18. Control and radiation processed (at optimum dose) RTC products for ash

gourd, drumstick and pumpkin at the end of intended storage period.

For ash gourd, optimum solution obtained was a shelf life of 12 d at a radiation dose of 2

kGy, while for drumstick and pumpkin the optimum parameters were a shelf life of 12

and 21 d respectively at a radiation dose of 1 kGy. The RTC products developed for the

selected vegetables were further characterized for sensory quality by quantitative

descriptive analysis (QDA), nutritional quality in terms of radical scavenging activity,

total phenolic and flavonoid content, vitamin C content, head space gaseous composition

inside the packages and aroma quality at the optimized conditions.

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3.2.1. Sensory quality evaluation by Quantitative Descriptive Analysis (QDA)

The fresh control samples as well as those treated with optimum radiation dose and stored

for intended duration were evaluated for sensory attributes by QDA. Independent training

sessions were carried out to familiarize the sensory panel for assessing the product quality

and generate attributes for descriptive analysis.

Ash gourd: The sensory attributes evaluated for RTC ash gourd were texture, color,

aroma (ash gourd, irradiated, buttery and musty) and taste (ash gourd, irradiated). The

control samples at the end of storage (12 d) were not evaluated as they were spoiled. The

data obtained are represented by a spider diagram (Figure 19). The texture of the

processed product was not significantly affected after radiation processing on day zero;

however, softening was observed on the twelfth day of storage, which is in agreement

with the scores obtained on the hedonic scale. At the end of storage period (12 d), the

radiation processed product had slightly lower scores for attributes like buttery and ash

gourd aroma. No difference in taste was observed between processed products and fresh

control samples. QDA results thus indicate that radiation processed (2 kGy) ash gourd

had acceptable aroma, taste and textural properties during an extended storage period of

12 d.

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Figure 19. Quantitative descriptive analysis of fresh control and radiation-processed ash

gourd samples at day zero and twelve of storage.

Drumstick: The data obtained from QDA of RTC drumstick samples is represented as a

spider graph in Figure 20. The sensory attributes studied were texture, color (green,

brown), aroma (drumstick, green, irradiated, and musty) and taste (drumstick, sweet and

irradiated). Control samples on day 12 were not evaluated for taste as they exhibited

visible fungal growth. On day zero no significant effect of irradiation was observed on the

attributes studied. At the end of intended storage period (12 d), the irradiated samples

exhibited slightly lower but statistically insignificant scores for texture and color.

However, no significant effect of storage was seen in the other attributes of radiation

processed samples as compared to fresh-cut drumstick samples of day zero. The results

138

indicated that radiation treated (1 kGy) samples had acceptable sensorial properties

during an extended storage period of 12 d.


drumstick samples at day zero and twelve of storage.

Pumpkin: The sensory scores as obtained by QDA of pumpkin are shown pictorially by a

spider graph in Figure 21. Control samples on day 21 were not evaluated for taste as they

were spoiled. After 21 d of storage, the irradiated (1 kGy) product had marginally lower

scores for texture and color. This is also evident from the instrumental data for color and

texture. However, no significant difference in taste and aroma were noted between

radiation-treated products at day 21 and fresh control samples. No irradiated off flavor or

taste was perceived by the assessors. Similar results have been reported in various

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radiation-treated minimally processed vegetables [86]. QDA results thus established that

radiation processed (1 kGy) pumpkin had acceptable aroma, taste and textural properties

during an extended storage period of 21 d.


pumpkin samples at day zero and 21 of storage.

3.2.2. Nutritional quality

The fresh-cut products are claimed to be convenient and healthy alternatives to fulfill the

dietary needs for fresh food. However, the changes that happen during harvesting,

handling and processing can affect antioxidant status. Lindley (1998) [163] and Klein

(1987) [164] reviewed the nutritional consequences of minimal processing on fruit and

vegetables and concluded that conditions that maintain desirable sensory characteristics

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will also preserve nutrients. Levels of ascorbic acid, carotenoids or polyphenols can

reflect the variations in antioxidant capacity of fruit and vegetables [164]. The antioxidant

potential in the form DPPH radical scavenging activity, and the principal components

which could contribute towards the antioxidant potential such as phenolic, flavonoid and

vitamin C contents were therefore analyzed in response to radiation treatment during

storage.

3.2.2.1. DPPH Radical scavenging activity

Ash gourd: Total antioxidant activity of ash gourd was found to be 169.9 ± 11.3 mg kg−1

of fresh weight. In control samples, the radical scavenging activity decreased on day 5

and then remained unchanged on subsequent storage. The activity increased significantly

(p ≤ 0.05) in response to radiation treatment compared to the corresponding control at all

stages of storage (Figure 22 A). A lowering in antioxidant activity was, however, noted

on storage of samples beyond 8 d in the irradiated samples.

Drumstick: The radical scavenging activity of drumstick was found to be 125.56 ± 8.6

mg kg−1

of fresh weight. Radiation treatment increased the antioxidant potential compared

to control samples. This increase was, however, not statistically significant (p ≤ 0.05). An

appreciable decrease in the activity was observed during storage in control as well as

irradiated samples (Figure 22 B). A decrease of 39 % and 43 % in activity was observed

in control and radiation processed samples on day 5. Beyond 5 d, activity decreased

continuously in control samples, while it remained unaffected in radiation processed

samples. The radiation treated samples (1 kGy) were therefore better than the

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corresponding control in terms of DPPH scavenging activity at the end of storage period

(12 d).

Figure 22: Variation in DPPH radical scavenging capacity of control and radiation

processed RTC vegetables with storage. (A) Ash gourd, (B) Drumstick, (C) Pumpkin

Pumpkin: An appreciable radical scavenging activity in pumpkin has been reported

earlier [165]. In the present study, the antioxidant capacity in pumpkin was found to be 72

mg/kg of pumpkin. No statistically significant (p < 0.05) change was observed in

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antioxidant activity due to radiation processing (1 kGy) at zero day (Figure 22 C). During

storage, the antioxidant activity was unchanged in control samples up to 7 days, while a

20% decrease was observed on day 21. On the other hand, irradiated samples had no

significant (p<0.05) change in the activity during the intended storage period of 21 d.

Various studies are available in literature reporting changes in the antioxidant potential of

fresh-cut vegetables as a result of processing. A decrease in the antioxidant activity after

processing is reported for fresh-cut spinach [166] and mandarin [167], while, wounding is

known to increase the antioxidant activity of Iceberg and Romaine lettuce [168]. It is

known that irradiation generates free radicals that may act as stress signals and trigger

stress responses in vegetables, resulting in increased antioxidant synthesis [94]. It has

been also proposed that gamma radiation is capable of breaking the chemical bonds of

polyphenols, resulting in the release of low molecular weight soluble phenols, leading to

an increase in antioxidant rich phenolics [92]. These could possibly be some of the

reasons for observed increase in radical scavenging activity in irradiated ash gourd and

drumstick. However, during storage, the activity declined which might be due to

degradation of antioxidants as a result of oxidation on prolonged storage.

3.2.2.2. Total phenolic and flavonoid content

Phenolic compounds including flavonoids are important bioactive compounds that have

the ability to reduce free radical formation and to scavenge free radicals. They act in

plants as antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellents,

and light screener [43]. The total phenolic and flavonoid content as estimated in ash

gourd, drumstick and pumpkin is shown graphically in Figure 23.

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Figure 23: Variation in total phenolic and flavonoid content in ash gourd (A & D);

drumstick (B & E) and pumpkin (C & F) due to radiation processing and storage.

Ash gourd: Total phenolic content was found to be 102.08 ± 3.44 mg kg−1

of ash gourd

in the present study. Changes in total phenolic and flavonoid content with radiation

processing and storage are depicted in Figure 23 A & D respectively. Radiation treatment

resulted in a significant (p ≤ 0.05) increase in the phenolic and flavonoid content on day

zero, and it remained unaffected during the entire storage period. In control samples, the

total phenolic content decreased significantly (p ≤ 0.05) during an initial storage period of

5 d, while a marginal but statistically insignificant increase was observed in the flavonoid

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content on day 5. The phenolic as well as flavonoid contents remained unchanged during

the remaining storage period. Total phenolic and flavonoid contents in radiation treated

samples were found to be significantly higher (p < 0.05) than the corresponding controls

on all storage days studied. Thus the nutritional quality of the developed radiation

processed product was better at the end of storage period (12 d).

Drumstick: A significant effect of radiation processing was observed on the total

phenolic content of RTC drumstick (Figure 23 B). The phenolic content in the radiation

processed samples (24 ± 2.35 mg/kg) was twice that of that of the control (11.24 ± 0.69

mg/kg) on day zero, which remained unchanged during storage duration. Irradiated

samples exhibited significantly (p < 0.05) higher phenolic content than corresponding

controls on all days of storage.

At day zero, radiation processing caused a marginal increase in flavonoid content,

however, the increase was not statistically significant (Figure 23 E). A decrease in the

content was observed during storage in control as well as radiation processed samples.

The contents in control and irradiated samples were comparable at the end of storage

period (12 d).

Pumpkin: In contrast to the ash gourd and drumstick, a significant (p < 0.05) decrease in

the total phenolic and flavonoid content was observed in response to radiation treatment

(Figure 23 C & F). Gamma radiation and UV-C induced decrease in antioxidant

compounds such as phenolics and flavonoids has been reported previously in various food

commodities [169, 170]. During storage, the contents decreased in control samples,

however it was unchanged in the radiation processed samples. A decrease in the flavonoid

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content during storage in orange and grapefruit juices has been reported earlier [171]. The

decrease was explained by degradation or oxidation of sensitive phenolic constituents

during extended storage.

Radiation induced increase in the flavonoid content was reported earlier in mushrooms

[172] and bitter gourd at radiation doses ranging from 0.25 – 1.0 kGy. Accumulation of

phenolic compounds and flavonoids in response to radiation as a defense mechanism in

plants has been reported. The increase has been attributed to the phenylalanine ammonia

lyase activity which is one of the key enzymes in the synthesis of phenolic compounds in

plant tissues [173]. Increase in phenolic compounds in irradiated plant produce has also

been attributed to depolymerization and dissolution of cell wall polysaccharides which

facilitated higher extractability [85]. The results obtained in the present study are in

agreement with those reported by other authors for minimally processed vegetables [173,

174]. The trends obtained for the variation in phenolic contents in response to radiation

treatment and storage were similar to that obtained for DPPH radical scavenging activity.

It indicates that phenolic compounds present in ash gourd, drumstick and pumpkin play

an important role towards their radical scavenging potential.

3.2.2.3. Vitamin C content

Vitamin C is one of the most sensitive vitamins, being degraded quickly on exposure to

heat, light, oxygen, processing and storage. It works as an antioxidant by protecting the

body against oxidative stress and also as a cofactor in several vital enzymatic reactions

[175]. It was therefore of interest to monitor the effect of radiation processing and storage

on the content of vitamin C in the developed RTC products.

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Figure 24. Effect of radiation processing and storage on ascorbic acid content of (A) Ash

gourd; (B) Drumstick; and (C) Pumpkin

Ash gourd: The amount of vitamin C in the present study was found to be 250 mg/kg of

ash gourd. The amount estimated is in agreement with the previous reports available [135,

100]. Vitamin C content of RTC ash gourd remained unaffected in response to radiation

treatment. No significant change (p ≤ 0.5) in the vitamin C content was observed during

storage in both the control (255–222 mg/kg) and radiation processed ash gourd (242–219

mg/kg) in the present study (Figure 24 A). Pandey et al. have, however, reported a

147

decreasing trend in the total vitamin C content in intact ash gourd during storage [176].

This different observation could be due to the methodology adopted wherein the total

ascorbate content was measured in its reduced form in the present study.

Drumstick: The content of vitamin C in fresh control drumstick samples (145.50 ± 9.17

mg/kg) was found to be in agreement with that reported in literature (141 mg/kg of edible

portion) [112]. A 13 % decrease in the content of vitamin C was observed immediately

after radiation treatment (1 kGy) (Figure 24 B). Storage resulted in a marginal decrease in

the vitamin C content of control as well as irradiated samples, however, this decrease was

not statistically significant (p < 0.05).

Pumpkin: Fresh control samples of pumpkin had vitamin C content of 600 mg/kg. A 27

% decline in vitamin C content was observed immediately after radiation processing (1

kGy), which did not change significantly (p < 0.05) during storage (Figure 24 C).

However, in the control pumpkin samples, a 60 % decrease in vitamin C content was

observed on day 21. At the end of the storage period, the vitamin C content of radiation-

treated samples (0.35 mg/kg) was significantly higher (p<0.05) than that of control

samples (0.23 mg/kg), making its nutritional quality better than control samples on day

21.

Losses in vitamin C content during storage have been known in different fruits and

vegetables [177]. Better retention of vitamin C during extended storage in radiation

processed sample has been observed earlier in different vegetables [12]. Thus, results

obtained in the present study are in agreement with the previous reports available in

literature.

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3.2.3. Headspace gaseous composition

Minimally processed vegetables are living tissues. Damaged plant tissues exhibit an

increase in respiratory rate. It has been reported that tissues with high respiratory rates

have shorter postharvest life [161]. Headspace gas composition inside the packages plays

an important role in controlling the nature and content of microbial growth. The content

of O2 and CO2 was therefore monitored for both the control and radiation processed

samples in all the three RTC vegetable packages at the optimized process parameters.

Ash gourd: Significant (p ≤ 0.05) effects of irradiation and storage time were observed

for both gases (O2 and CO2) in the packages (Figure 25A). In the control samples, a

continuous decrease in O2 and an increase in CO2 were observed during storage.

Irradiated samples had a higher CO2 concentration compared to the control till 3 d of

storage. During storage, the CO2 concentration inside the package decreased gradually,

while a corresponding increase in O2 concentration was observed in the radiation

processed samples until the fifth day. However, from day five onwards it remained

constant for the intended duration of storage. At the end of storage, the gaseous

composition was 13 % O2 and 8.8 % CO2 for control, and 19 % O2 and 2.8 % CO2 for the

irradiated samples.

149

Figure 25. Headspace composition in control and radiation processed RTC (A) Ash gourd

(B) Drumstick and (C) Pumpkin packages during storage.

Drumstick: O2 and CO2 contents inside the packages are depicted in Figure 25 B.

Radiation processing resulted in significant (p < 0.05) decrease in O2 and increase in CO2

content immediately after treatment. The CO2 content of radiation processed samples was

higher than the control till day 7. However, during further storage, the CO2 content

150

decreased in radiation processed samples and then remained constant, achieving

equilibrium with the atmosphere. At the end of storage period (12 d), the gaseous

composition for control and irradiated samples was 18.28 % O2, 3.69 CO2 and 19.5 % O2,

2.39 % CO2 respectively.

Pumpkin: Immediately after radiation treatment, packages of irradiated pumpkin had

significantly (p < 0.05) lower O2 and higher CO2 levels (12.91% O2 and 8.59% CO2) than the

controls (17.88 % O2 and 3.62% CO2) as depicted in figure 25 C. After 7 days of storage O2

and CO2 content in packages subjected to radiation processing were 19 and 2.5 %,

respectively. Thus an increase in oxygen concentration as compared to zero day was

observed, which remained constant for the remaining storage duration.

The continuous decrease in O2 and increase in CO2 observed during storage in control

samples could be due to product respiration. A similar effect was observed in the case of

cantaloupe and honey dew subjected to passive MAP [178]. Radiation induced decrease in

O2 and increase in the content of CO2 was noted in all the three products immediately after

radiation treatment. A similar effect has been demonstrated in broccoli and mushrooms

where increase in metabolic activity was cited as the reason for increased respiration rate

during the first 24 h following irradiation [12, 179]. However, the content of O2 increased

gradually on further storage. Such an observation was also reported in mushrooms subjected

to gamma irradiation were linked to the reduction in metabolic activity during storage [179].

After a certain storage period (5 d for ash gourd, 5 d for drumstick and 7 d for pumpkin), no

significant (p < 0.05) change in the O2 content was observed, indicating attainment of

equilibrium between package headspace and atmosphere. O2 content less than 2% in

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package headspace can result in anaerobic conditions leading to generation of off flavors and

possible growth of pathogens like Clostridium botulinum [180]. In the present study, the O2

concentrations observed for the processed samples during storage were between 16 to 19 %,

thus maintaining aerobic conditions within the package. Processing and packaging

conditions chosen in the present study were therefore suitable for the RTC products of the

selected vegetables.

3.2.4. Aroma quality - chemical analysis of free and bound aroma compounds and

identification of key odorants

Aroma is an important quality parameter that decides consumer choice and acceptability

of food. It is contributed by volatile compounds that are generally hydrophobic, and

usually occur in trace levels (ppm or ppb). These constituents show considerable variation

in both the nature and concentration in different food stuff. The nature of the food matrix

affects the concentration of volatile composition in the headspace and consequently their

perception by the consumer. In fresh food, flavor results from natural compounds

produced by enzymatic degradation during harvesting and processing [181]. Moreover,

the flavor of cooked food is due to numerous chemical and enzymatic reactions that are

influenced by temperature. Despite the presence of several volatile aroma compounds in a

food matrix, not all of them are responsible for the characteristic odor of a food product.

It is known that only a small portion of the large number of volatiles occurring in a food

matrix contributes to its overall perceived odor [182]. Further, these molecules do not

contribute equally to the aromatic profile of a sample. Sometimes, one substance alone is

152

able to reflect the approximate flavor of a product and, in this case, it is called “impact

compound”. But, in some circumstances, it is the combination of substances that

collectively interacts with the receptors from the nasal mucosa and is interpreted by the

brain to create a sensory impression typical for each product [182]. In general, the sensory

importance of an odor-active compound depends on its concentration in the matrix, and

on the limit of detection by human nose. Therefore, there is a great interest to determine

the contribution of various constituents in aroma isolate towards the overall flavor of a

product.

Intensive research carried out over the past three decades has demonstrated that, besides

the volatile constituents that contribute to odor, several aroma compounds accumulate as

non-volatile and flavorless glyco-conjugates, which make up a reserve of aroma to be

exploited [183]. These glyco-conjugates are shown to contribute finer notes to food.

Although odorless, they are able to release free aroma compounds by enzymatic or

chemical hydrolysis during processing and storage [39]. In the past few years, analysis of

flavor precursors in fruits, vegetables and spices have received increased attention. These

precursors in many cases have been shown to be more abundant than the free form to the

extent of 70-90 %. However, very few reports exist on the nature of the glycosidic

precursors in the vegetables.

Despite the fact that vegetables impart characteristic flavor (aroma, taste and color) to the

cuisine, very few reports exist on the nature of their aroma compounds. In the green form,

aroma of majority of the vegetables is indistinguishable from each other. Cooking results

in liberation of their characteristic aroma from their precursors. The nature of these

153

precursors has been exhaustively investigated in the vegetables of the Cruciferae family,

where in liberation of aroma from glucosinolates has been reported. No report, however,

exists on the nature of these precursors in majority of the vegetables, specifically of the

Indian origin. To the best of our knowledge, only one report exists on the nature of free

aroma compounds in ash gourd and pumpkin, while the drumstick aroma has not been

explored so far [106, 184]. The glycosidic aroma precursors in these vegetables have not

been studied till date. No study exists on the aroma impact compounds that contribute to

the characteristic odor of these vegetables. It was therefore of interest to investigate free

and bound aroma compounds of these vegetables and characterize the key odorants.

3.2.4.1. Ash gourd

3.2.4.1.1. Free aroma analysis: The free aroma of ash gourd was extracted employing

three different techniques – simultaneous distillation extraction (SDE), high vacuum

distillation (HVD) and solid phase micro extraction (SPME). The detailed methodology

adopted has been described in section 2.2.2.1.1. Although a widely used technique, SDE

has the disadvantages that some of the heat labile constituents may undergo degradation,

or the low boiling volatiles may be lost at the temperatures employed [185]. A

characteristic cooked odour is also perceived in many cases. Milder techniques such as

high vacuum distillation (HVD) employing distillation under reduced pressure and SPME

have therefore been attempted to obtain isolate that represent truer aroma [130]. The

aroma volatiles were thus extracted using SDE, HVD and SPME in order to ascertain

generation of artefacts as gas chromatographic (GC) patterns are largely influenced by

extraction procedures [186]. The GC/MS chromatograms obtained for SDE, HVD and

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SPME are presented in Figure 26. Table 27 provides a quantitative distribution of the

constituents identified in the essential oils isolated by SDE, HVD as well as by SPME.

Figure 26: GC/MS profiles of volatile aroma of ash gourd obtained using SDE, HVD and

SPME methods.

Table 27. Quantitative distribution (µg/kg of ash gourd) of volatile oil components

identified in isolates obtained from SDE, HVD and SPME.

Compound KI HVD (µg/kg) SDE (µg/kg) SPME (µg/kg)

Alcohols

1-Butanol 655 1.46 ± 0.15

155

2-Pentanol 701 0.41 ± 0.05

3-Methyl-1-butanol 735 1.29 ± 0.22

2-Methyl-1-butanol 742 0.31 ± 0.04

2,3-Butandiol 765 0.73 ± 0.19

2,4-Dimethyl-3-pentanol 825 0.1 ± 0.03

1-Hexanol 868 0.098 ±0.04 0.51 ± 0.15

1-Heptanol 965 0.012 ± 0.003 1.72 ± 0.02

2-Ethylhexanol 1021 0.068 ± 0.008 1.25 ± 0.43

Nonanol 1170 0.005 ± 0.003 0.33 ± 0.02

2-Decyn-1-ol 1275 0.15 ± 0.03

Aldehydes and ketones

3-Methylbutanal 640 1.08 ± 1.06

2-Pentanone 675 1.09 ± 0.06

2-Ethylbutanal 742 0.43 ± 0.17

4-Methyl-3-penten-2-one 795 1.03 ± 0.37

Acetoin 812 7.59 ± 1.15 7.62 ± 1.18 12.16 ± 1.72

Hexanal 815 16.42 ± 2.28

2E-Hexenal 854 0.006 ± 0.001 0.37 ± 0.14

Heptanal 902 0.11 ± 0.04

6-Methyl-5-hepten-2-one 985 0.47 ± 0.17

2-Octanone 988 0.011 ± 0.009 0.61 ± 0.19

Octanal 994 0.006 ± 0.003 0.71 ± 0.08

156

Nonanal 1093 0.024 ± 0.006 1.38 ± 0.16

Decanal 1209 0.003 ± 0.001 0.11 ± 0.03

Terpenes

β.-Pinene 970 0.35 ± 0.01

Sabinene 976 0.51 ± 0.09

Indene 1025 0.36 ± 0.04

l-Limonene 1032 0.47 ± 0.17

α-Terpineol 1185 0.003 ± 0.001 0.21 ± 0.04

Nerolidol 1550 0.014 ± 0.009

Others

Methyl Butyrate 735 0.3 ± 0.07

Ethyl acetate 806 0.014 ± 0.006 0.039 ± 0.006 53.94 ± 5.46

n-Butyl acetate 815 0.36 ± 0.11

3-Methyl butyl acetate 875 0.4 ± 0.06

Methyl hexanoate 915 0.22 ± 0.06

3E-Hexenoic acid 1010 0.2 ± 0.04

Verbenyl acetate 1255 0.09 ± 0.01

Data are expressed as mean ± standard deviation (n = 3).

Irrespective of the extraction techniques used, acetoin was found to be the major

constituent identified. It is proposed to originate from a side reaction during the

biogenesis of valine and leucine [187]. Ethyl acetate was the only other constituent

detected in all the three extracts. Unlike SDE and SPME, however, the GLC profile of

157

HVD isolates was characterized by a negligible content of aliphatic C6-C10 alcohols and

aldehydes such as hexanol, heptanol, 2-ethylhexanol, hexanal, heptanal, and octanal. The

C5, C6, and C8 alcohols and carbonyl compounds, responsible for green odour of several

vegetables, are known to be derived from linoleic and linolenic acids [188].

The GC/MS profile of SPME sample was characterized by both, a higher number of

compounds and a significantly higher content of individual constituents as compared to

that obtained from SDE and HVD. A total of 37 volatile compounds were detected in the

headspace-solid phase microextraction (HS-SPME) of ash gourd. Alcohols (10),

carbonyls (13), terpenes (6) and fatty acid esters (7) were the principal chemical classes

identified in HS-SPME. Major compounds detected in SPME, that were not present in the

other two extraction techniques were: 1-butanol (1.46 µg/kg), 2,3-butanediol (0.73

µg/kg), 3-methyl-1-butanol (1.29 µg/kg), 4-methyl-4-penten-2-one (1.03 µg/kg), 3-

methylbutanal (1.08 µg/kg), hexanal (16.42 µg/kg), 6-methyl-5-hepten-2-one (0.47

µg/kg), sabinene (0.51 µg/kg), limonene (0.47 µg/kg) and indene (0.36 µg/kg). The

volatile profile of ash gourd comprises a large proportion of esters of aliphatic acids

(Table 27). Use of SPME resulted in the detection of a large number of low molecular

weight esters, such as methyl butyrate (0.3 µg/kg), n-butyl acetate (0.36 µg/kg), 3-methyl

butyl acetate (0.4 µg/kg), methyl hexanoate (0.22 µg/kg) and trans-verbenyl acetate (0.09

µg/kg). These observations indicated that SPME was more efficient for the extraction of

volatile aroma compounds from ash gourd. Higher extraction of alcohols and esters by

SPME in comparison to SDE has been previously reported in volatile profile of beans

158

[181]. The evaporation step during SDE may increase the loss of the most volatile

compounds [181].

In an earlier study conducted by Wu et al. on volatile compounds of ash gourd, they

identified E-2-hexenal (green fruity, pungent vegetable like odour), n-hexanal (fatty green

grassy) and 3-methyl-1-butanol (fruity, alcoholic) as the major volatile constituents of this

vegetable [106]. These compounds were also found as major aroma volatiles in the

present study. However, other volatile constituents such as n-hexyl formate, 1-octen-3-ol,

undecen-1-ol, 2-hexyl furan, deca-2, 4-dienal, isoamyl alcohol and 2-heptenal earlier

reported by Wu et al. were not detected by us in this study [106]. It is interesting to note

that despite a high content of acetoin detected in the essential oil in our study this

compound was not earlier reported even in trace amounts as a constituent of the ash gourd

volatiles. The variation observed in aroma composition could be due to the difference in

variety and geographical origin of the vegetable studied.

3.2.4.1.2. Bound aroma analysis

The bound aroma glycosides were extracted using two different methods – XAD and

solid phase extraction (SPE) using C18 cartridges (section 2.2.2.2.2.). The use of XAD as

well as SPE is widely reported in literature [189]. The extracts obtained from XAD and

SPE were separately subjected to acid and enzymatic hydrolysis. The released free

aglycones were then subjected to GC/MS analysis. A higher number of peaks were

detected in the GC profile of enzymatically hydrolyzed glycosidic isolate. This could

possibly be due to mild conditions during enzymatic hydrolysis unlike the harsh

conditions that prevail during acid hydrolysis resulting in significant modification of the

159

released aglycones [133]. This is in accordance with the earlier reports on bound aroma

precursors in wine wherein enzymatic hydrolysis are known to represent the true aroma

profile (without further chemical transformations) [190]. Enzymatic hydrolysis as a method

for release of free aglycones from glycosidically bound aroma precursors was thus adapted

for further studies.

Figure 27. GC/MS profile of free aglycones released from bound aroma precursors of ash

gourd obtained from XAD and SPE.

The representative GC/MS chromatograms of the released aglycones obtained after

enzymatic hydrolysis of XAD and SPE isolates are shown in Figure 27. As evident from the

chromatograms, significantly higher number of peaks was observed in the SPE extract. Table

28 provides a quantitative distribution of aglycones liberated from glycosidic precursors

extracted by XAD and SPE. Acetoin was detected as the major aglycone released after

hydrolysis of bound aroma glyco-conjugates isolated by both the methods. Acetoin

accounted for 98% of the bound aroma compounds in XAD extract and 46% in the SPME

160

isolate. The occurrence of acetoin as a major glyco-conjugate further confirms this

compound as an important aroma constituent of ash gourd. Among the other bound aroma

constituents identified in XAD extract, 2-pentanol (minty), 2-ethyl-1-hexanol (floral), 3-

methyl butanol (ripe onion) and vanillin (vanilla like) with low odor thresholds may also

contribute to the overall odor of the vegetable.

Table 28. Quantitative distribution (µg/kg ash gourd) of free volatile components

obtained from glycosidic precursors extracted by XAD and SPE.

Compound KI XAD (µg/kg) SPE (µg/kg)

Alcohols

1-Butanol 655 6.75 ± 0.26

3-Methyl-1-butanol 735 0.17 ± 0.03 23.48 ± 1.58

2-Methyl-1-butanol 742 12.31 ± 0.05

2,3-Butandiol 765 5.86 ± 0.05

1,3-Butanediol 784 0.08 ± 0.02 5.89 ± 0.05

1-Hexanol 868 3.09 ± 0.03

2,4-Hexadien-1-ol 875 0.79 ± 0.02

2-Pentanol 959 0.09 ± 0.02 2.24 ± 0.06

1-Heptanol 965 1.51 ± 0.07

2-Ethylhexanol 1021 1.68 ± 0.03 10.65 ± 0.92

4-Penten-1-ol 1072 Tr

1-Nonanol 1171 1.17 ± 0.17

Trans-2-undecen-1-ol 1368 0.17 ± 0.03

161

Dodecanol 1473 0.16 ± 0.01

Hexadecanol 1879 Tr

Octadeca-9,12-dien-1-ol 2035 0.17 ± 0.012

Cis-9-octadecen-1-ol 2082 0.93 ± 0.12


3-Methyl-butanal 640 0.14 ± 0.04

2-Pentanone 675 3.23 ± 0.24



Acetoin 812 826 ± 3.456 138.64 ± 10.02

Hexanal 815 12.74 ± 1.18

4-Hydroxy-2-butanone 820 Tr

2-Hexenal 854 2.19 ± 0.13

Heptanal 902 2.66 ± 0.16

Benzaldehyde 961 Tr


2-Octanone 988 4.89 ± 0.77

Octanal 994 1.34 ± 0.35

Nonanal 1093 Tr 2.36 ± 0.41

Decanal 1193 Tr 0.67 ± 0.25

3-Hydroxy-4-methoxy

benzaldehyde

1391 1.01 0.581

162

Dodecanal 1407 0.505 ± 0.062

Terpenes

Myrcene 955 0.8 ± 0.59

β-Pinene 970 2.28 ± 0.49

Sabinene 976 2.34 ± 0.1

Indene 1025 2.98 ± 0.15

l-Limonene 1032 5.8 ± 0.11

Cymene 1113 0.69 ± 0.18

Allo-ocimene 1125 1.11 ± 0.09

α-Terpineol 1185 1.82 ± 0.08

Others

Methyl butyrate 735 1.45 ± 0.05


Isobutyl propionate 860 2.28 ± 0.2

3-Methyl butyl acetate 875 2.34 ± 0.1

Methyl hexanoate 920 2.29 ± 0.16

Nonanoic acid 1278 1.61 ± 0.04

Methyl decanoate 1325 1.5 ± 0.08

Decanoic acid 1372 1.59 ± 0.06

Dodecanoic acid 1565 1.86 ± 0.07

Ethyl palmitate 1990 1.69 ± 0.03

Data are expressed as mean ± standard deviation (n = 3); Tr – Detected in traces

163

The XAD extract of ash gourd was characterized by the presence of 10 alcohols and 7

carbonyl compounds (5 aldehydes and 2 ketones). The major alcohols identified were 3-

methyl-1-butanol (0.17 µg/kg), 2-ethyl hexanol (1.68 µg/kg), dodecanol (0.16 µg/kg),

trans-2-undecen-1-ol (0.17 µg/kg), octadeca-9,12-dien-1-ol (0.17 µg/kg) and cis-9-

octedecen-1-ol (0.93 µg/kg). As in free aroma isolate, acetoin (826 µg/kg) was also

identified as the most abundant bound precursor in the XAD isolate. The other aldehydes

detected in comparatively lower amounts include 4-hydroxy-2-butanone, benzaldehyde,

3-hydroxy-4-methoxy benzaldehyde, nonanal, decanal and dodecanal.

SPE isolate on the other hand, was characterized by the presence of total 42 compounds

with 11 alcohols, 13 carbonyl compounds, 8 terpenes and 10 fatty acids or esters. The

major compounds identified could be classified as alcohols: 3-methyl-1-butanol (23.48

µg/kg), 2-methyl-1-butanol (12.31 µg/kg), 2-ethyl hexanol (10.65 µg/kg); carbonyl

compounds: acetoin (138.64 µg/kg), 4-methyl-3-penten-2-one (25 µg/kg), hexanal (12.74

µg/kg) and terpenoids: limonene (5.8 µg/kg), β-pinene (2.28 µg/kg), sabinene (2.34

µg/kg) & indene (2.98 µg/kg). Apart from the compounds which were identified in XAD

extract, alcohols such as butanol, 1-nonanol, 2, 3-butanediol, 2-methyl-1-butanol,

hexanol, heptanol and carbonyl compounds including 4-methyl-3-penten-2-one, 2-

pentanone, 2-hexenal, heptanal, 6-methyl-5-hepten-2-one, 2-octanone were the other

major compounds identified in the SPE extract. Also certain terpenes such as β-pinene,

sabinene, myrcene, cymene, limonene, indene and α-terpineol were identified only in the

SPE extract. The presence of glycosidic precursors of terpenes has been widely reported

in literature [191]. Some higher aldehydes and alcohols such as dodecanal, dodecanol,

164

hexadecanol, octadeca-9,12-dien-1-ol, cis-9-octadecen-1-ol, which were present in XAD

extract were not detected in the SPE extract. However, these high molecular weight

compounds with high boiling points and odor threshold values, may not contribute

significantly to the overall aroma of the vegetable. The above results further confirm SPE

as an efficient technique for extraction of bound aroma glycosides from ash gourd,

confirming earlier reports on the suitability of SPE for extraction of aroma glycosides

from food stuffs [192].

3.2.4.1.3. Identification of key odorants

Aroma active compounds play a major role in imparting characteristic odor of a food.

Among the several compounds that make up the aroma isolate, a single or a very few

compounds in combination are known to contribute to its characteristic odor. The

presence of acetoin in considerable amount, both in free and in bound form suggests its

possible role as an aroma active compound of the vegetable. The study was thus focused

further on the identification of the most potent odorants of ash gourd. GC–O method

based on detection frequency (Global Olfactometry Analysis) of odors was employed as

the method was rapid and required no trained panelist. Eight odor active compounds were

sniffed by at least more than four assessors during olfactometric analysis of volatiles from

SDE and SPME that were injected separately on a DB-5 column. Table 29 presents odor

descriptors perceived by the panel for the aroma constituents in order of elution in this

column. A variety of odor qualities such as cut ash gourd, green, herbaceous, fatty green

were perceived. The nine judges constituting the sensory panel agreed that the odour of

the extract was typical buttery green vegetable like. Two regions at Rt 4.5–5.5 min and

165

17.5–19.0 min corresponding to the cut ash gourd and fatty green, respectively, closely

resembled the odor of the vegetable.

Table 29. ODP analysis of ash gourd SDE oil

Rt (min) Aroma perceived KI Compound

identified

OAV

4.0-4.5 Fruity 806 Ethyl acetate 5.8

4.8 – 5.67 Buttery 812 Acetoin 9522.5

9.5-9.7 Green-fruity/vegetable like 854 2E-Hexenal 352.9

10.00 Green flowery 868 Hexanol 39.2

13.75-14.00 Earthy/ herbaceous 965 Heptanol 4000

16.85-17.15 Fruity 988 2-octanone 220

17.53-17.65 Fatty green 994 Octanal 8571

17.75-17.95 Floral 1021 2-Ethyl hexanol ND

18.5-19.0 Green/fatty/soapy 1093 Nonanal 24000

21.35-21.54 Fatty/green 1170 Nonanol 100

21.90-22.05 Fruity/anise 1185 α-Terpineol 8.57

22.15-22.25 Green/nutty 1193 Decanal 1500

25.52-25.92 Dry wood/hay 1550 Nerolidol ND

ND – not detected

The odor detection frequency chromatogram of the volatile constituents analysed by GC–

O is shown in Figure 28. The DF data showed highest frequencies for acetoin (buttery/cut

ash gourd type), octanal (fatty green), nonanal (green/fatty/soapy) while somewhat lower

frequencies were found for 2-hexenal (green fruity/vegetable like), hexanol (green

166

flowery), heptanol (earthy/herbaceous), 2-octanone (fruity), nonanol (fatty/green) and

decanal (green/nutty). The identities of the eight compounds listed were established based

on their retention index, odour quality, and mass spectral data with those of standard

compounds. Among the odour active regions with highest DF, acetoin with a

characteristic creamy, fatty and buttery aroma, octanal with a fatty green odour and

nonanal with a green, fatty and soapy odour has not been reported earlier as an aroma

compound of this vegetable.

Acetoin is widely reported to occur in apples, asparagus, black currants, banana, litchi,

guava, blackberry, wheat, broccoli, brussel sprouts, cantaloupe, butter and yogurt [191,

193, 194]. It has also been proposed to be a major characteristic compound of lychee

aroma [191]. Alkenals and alkanals are known to contribute fatty-oily, slightly rancid

odours and are reported to be present in several vegetables [135]. Thus acetoin, octanal

and nonanal could be proposed as the most potent odorants of ash gourd. The latter two

compounds namely octanal and nonanal possess very low thresholds and thus were well

perceived during global analysis. Despite a high odour threshold, acetoin was also well

perceived by the sensory panel. This could possibly be due to the high concentration of

this compound in the vegetable. A high OAV value of this compound (Table 29), even

higher than octanal clearly suggests that its contribution to the overall odour of the

vegetable might be significant. Chemical structures and mass spectra for most potent

odorants of ash gourd are represented in Figure 29.

167

Figure 28. Odor Detection frequency chromatogram of Ash gourd SDE volatiles (Refer to

Table 29 for KI)

In order to further ascertain the role of acetoin as the odour active constituent of ash

gourd, the region corresponding to buttery/cut ash gourd odour, as perceived by the panel

was collected from the TCD vent and further subjected to GC/MS analysis. Acetoin was

the only constituent detected in the isolate (Figure 30), further confirming the role of this

compound as the key aroma active compound of the vegetable.

0

1

2

3

4

5

6

7

8

9

806 812 854 868 965 985 994 1021 1093 1170 1185 1193 1550

DF

KI

168

Figure 29. Chemical structures and mass spectra for most potent odorants of ash gourd

Figure 30: GC/MS chromatogram of cryotrapped fraction (4.5-5.5 min) isolated

corresponding to ash gourd aroma.

169

3.2.4.2. Drumstick

3.2.4.2.1. Free aroma analysis

The free aroma of drumstick was extracted employing SDE and SPME (section

2.2.2.2.1). The representative GC chromatograms obtained are shown in Figure 31, while

the quantitative distribution of the constituents identified is presented in Table 30.

Figure 31. GC/MS profiles of volatile aroma compounds of drumstick obtained using

SDE and SPME methods.

170

Table 30. Quantitative distribution (µg/kg of drumstick) of volatile oil components

identified in SDE and SPME.

Compound KI SDE (µg/kg) SPME (µg/kg)

Alcohols

Ethanol < 600 16.1 ± 2.08

1-PENTEN-3-OL 685 9.2 ± 0.13

1-Butanol, 3-methyl 735 2.12 ± 0.13 4.89 ± 1.48

1-Butanol, 2-methyl 742 4.41 ± 1.59

2-Penten-1-ol, (Z) 767 2.71 ± 0.29

2-Hexen-1-ol, (E) 853 3.09 ± 0.22

3-Hexen-1-ol, (Z) 857 0.016 ± 0.03 28.72 ± 3.99

1-Hexanol 868 0.02 ± 0.01 4.89 ± 0.22

1-Heptanol 965 0.63 ± 0.37

1-Octen 3 ol 985 0.18 ± 0.02

1,8-Octanediol 995 0.042 ± 0.01

2-Ethyl hexanol 1021 7.66 ± 1.86

1-Nonanol 1171 0.55 ± 0.35

2-Decen-1-ol 1271 0.78 ± 0.19

1-Decanol 1275 8.94 ± 2.34


Acetaldehyde < 600 12.17 ± 3.29

Iso butyraldehyde 615 14.46 ± 3.1

171

Butanal, 3-methyl 640 21.75 ± 1.79

1-Penten-3-one 680 10.98 ± 3.26

Pentanal (CAS) 697 5.83 ± 1.98

2-Pentenal, (E) 745 4.83 ± 0.26

3-Hydroxy-2-butanone 812 9.25 ± 0.21 21.84 ± 2.69

Hexanal 815 63.6 ± 8.38

Trans-2-hexenal 854 136.21 ± 13.53

Heptanal 899 0.88 ± 0.26

4-Heptenal, (Z) 902 0.18 ± 0.03

2-Heptenal 955 0.22 ± 0.01

Benzaldehyde 961 2.42 ± 0.14

2-Octanone 988 2.23 ± 0.52

Benzeneacetaldehyde 1043 2.34 ± 1.08

2-Octenal, (E) 1060 0.004 ± 0.001 0.56 ± 0.06

Nonanal 1093 3.44 ± 0.33

2,6-Nonadienal, (E,Z) 1154 6.08 ± 3

Decanal 1193 14.42 ± 2.15 21.05 ± 4.52

Dodecanal 1407 0.004 ± 0.001 30.99 ± 8.73

2-Decenal 1261 2.06 ± 0.05 6.28 ± 1.55

2,4-Decadienal 1315 2.2 ± 0.33

Nitrogen and sulphur containing compounds

Propanenitrile, 2-methyl 625 12.16 ± 1.73

172

Butanenitrile, 2-methyl 695 3.43 ± 0.86

Propanesulfonylacetonitrile 1250 28.42 ± 8.07

2-hydroxy propane nitrile 725 8.54 ± 0.87

Isopropyl isothiocyanate 835 109.99 ± 17.66

Dimethyl sulfone 920 0.029 ± 0.001

Butane, 2-isothiocyanato 946 5.14 ± 0.15

Isobutyl isothiocyanate 974 14.79 ± 0.49

Benzyl nitrile 1138 0.004 ± 0.001

Benzene isonitrile 1198 1.86 ± 0.22

2-methoxy-3-(isobutyl)-pyrazine 1204 0.005 ± 0.001

Benzothiazole 1215 10.3 ± 1.3 19.25 ± 3.87

Terpenes and others

Ethyl acetate 806 8.95 ± 1.65

Z-4-Hexenyl acetate 1012 0.016 ± 0.002

l-LIMONENE 1031 2.52 ± 1.31

Trans-Linalool oxide 1088 2.1 ± 0.12

Linalool 1098 0.004 ± 0.001 0.59 ± 0.36

α-Terpineol 1185 12.28 ± 0.24


As in ash gourd, use of SPME also resulted in isolation of a higher number of aroma

compounds in drumstick. The evaporation step during SDE could possibly have resulted

in the loss of the most volatile compounds [181]. The GLC profile obtained from SPME

173

was dominated by alcohols, carbonyls and esters. A higher extractability of alcohols and

esters by SPME fibre used has been reported earlier in beans by Barra et al. [181]. This

could possibly explain the dominance of above class of compounds in the volatile profile.

The GLC profile obtained from SDE extract was characterized by the presence of 17

volatile compounds of which one terpene, 4 alcohols, 5 carbonyl compounds, 5 nitrogen

or sulfur containing compounds and two esters were identified. The major aroma

compounds present in the SDE isolate were 3-methyl-1-butanol (2.12 µg/kg), 3-hydroxy-

2-butanone (9.25 µg/kg), 2-hydroxy propane nitrile (8.54 µg/kg) and ethyl acetate (8.95

µg/kg).

SPME profile, on the other hand, was characterized by the presence of 48 volatile

compounds. It included 14 alcohols, 22 carbonyl compounds, 8 nitrogen and sulphur

containing compounds and 4 terpenes. The major alcohol and carbonyl compounds

identified were 3-hexen-1-ol (28.72 µg/kg), 2-ethyl hexanol (7.66 µg/kg), decanol (8.94

µg/kg), 3-hydroxy-2-butanone (21.84 µg/kg), 3-methyl butanal (21.75 µg/kg), 1-penten-

3-one (10.98 µg/kg), trans-2-hexenal (136.21 µg/kg) and dodecanal (30.99 µg/kg).

Interestingly, the SPME profile of drumstick was characterized by the presence of

various nitrogen and sulphur containing compounds. It included nitriles: 2-methyl

propane nitrile (12.16 µg/kg), 2-methyl butane nitrile (3.43 µg/kg), propane sulfonyl

acetonitrile (28.42 µg/kg), benzene isonitrile (1.86 µg/kg); isothiocyanates: isopropyl

isothiocyanate (109.99 µg/kg), isobutyl isothiocyanate (14.79 µg/kg); and benzothiazole

(19.25 µg/kg). Nitriles, isonitriles and isothiocyanates are widely reported to be present in

vegetables of order Brasicales and are generated by the hydrolytic breakdown of their

174

precursor glucosinolates. These hydrolysis products are known to be responsible for the

characteristic aroma of various vegetables of this order [58]. 2-hydroxy propane nitrile,

dimethyl sufone and 2-methoxy-3-isobutyl pyrazine which were present in the SDE

isolate, were not detected in the profile obtained from SPME. These could have been

generated as a consequence of heat treatment employed during SDE.

Despite the fact that drumstick fruits have a distinct and pleasant aroma, no studies on the

detailed aroma composition have been reported till date. The volatile aroma compounds

of drumstick leaves and flowers have, however, been exhaustively explored. In a study

conducted by Dev et al., effect of hot air drying on the volatiles of drumstick pods was

studied using z-Nose (an electronic nose) [195]. The effect of different drying methods -

microwave-assisted hot air drying (MAHD) and conventional hot air drying methods at

different temperatures were investigated by these authors. Among the various volatiles

detected, β-sitosterol was the major compound while 2,3-Dihydro-3,5-dihydroxy-6-

methyl-4H-pyran-4-one, n-hexadecanoic acid and 9,12-octadecadienoic acid were the

minor compounds (< 10 ng/g) identified by them using GC/MS. Microwave drying was

shown not to cause any significant variation in the content of these volatiles. However,

detailed composition of aroma volatiles was not studied by these researchers.


Figure 32 represents the GC/MS profiles of aglycones released after enzymatic hydrolysis

of the bound aroma glycosides of drumstick. The quantitative distribution of the

175

aglycones identified in XAD and SPE hydrolyzates of drumstick are presented in Table

31.

Figure 32. XAD and SPE chromatograms of drumstick

Table 31. Quantitative distribution (µg/kg drumstick) of free volatile components

obtained from glycosidic precursors extracted by XAD and SPE.

Name KI XAD (µg/kg) SPE (µg/kg)

Alcohols

Ethanol <600 10.88 ± 1.39

2-Butanol 605 36.64 ± 8.05

3-Pentanol 680 1.36 ± 1.07

2-Pentanol 690 0.18 ± 1.56

3-Penten-2-ol 710 1.88 ± 0.05

176

3-Buten-1-ol, 3-methyl- 725 1.02 ± 2.41

1,2-Propanediol 729 1.19 ± 2.02

1-Butanol, 3-methyl 735 12.11 ± 1.3

1-Butanol, 2-methyl 742 4.89 ± 0.14

2-Penten-1-ol, (Z) 767 0.16 ± 0.74

2-Hexanol (CAS) 780 0.77 ± 0.74

4-methyl-1-pentanol 835 0.1 ± 4.07

2-Hexen-1-ol, (E) 853 0.25 ± 6.03

3-Hexen-1-ol, (Z) 857 2.5 ± 0.97 24.35 ± 2.87

1-Hexanol 868 14.58 ± 0.04

1-Heptanol 965 1.3 ± 4.18

1-Octen-3-ol 985 0.48 ± 0.05

1-Hexanol, 2-ethyl- 1021 9.32 ± 0.03

Benzyl alcohol 1035 4.35 ± 1.07 7.34 ± 0.02

2-Octen-1-ol, (Z) 1060 0.19 ± 0.17

2-Methylphenol 1070 0.28 ± 0.13

Z-6-Nonenal 1097 1.02 ± 0.09

Benzene ethanol 1120 5.94 ± 1.05 2.06 ± 0.07

2E,6Z-Nonadien-1-ol 1158 0.5 ± 0.1

1-Nonanol 1171 1.02 ± 0.33

2-Propyl-1-heptanol 1190 3.06 ± 0.17

1-Octyn-3-ol 1225 1.42 ± 0.14

177

1-Decanol 1275 1.07 ± 0.61


Acetaldehyde <600 2.74 ± 0.24

Butanal, 3-methyl 640 1.18 ± 0.37


3-Hydroxy-2-butanone 812 14.31 ± 0.01

Hexanal 815 2.22 ± 2.46

2-Hexenal 854 2.09 ± 6.03

Heptanal 902 0.16 ± 4.07


Benzeneacetaldehyde 971 0.71 ± 1.98

5-Hepten-2-one, 6-methyl- 985 0.67 ± 0.31

2-Octanone 988 1.59 ± 13.12

Nonanal 1093 1.63 ± 4.18

Dodecanal 1407 0.75 ± 0.05

Nitrogen and sulphur containing compounds

Butanenitrile, 2-methyl 695 1.65 ± 0.01

Isopropyl isothiocyanate 835 0.19 ± 0.02

1-Propene, 3-isothiocyanato- 865 0.16 ± 0.13

Terpenes and others

1,3-Butadien-1-ol, acetate 785 0.04 ± 0.1

1-Butanol, 3-methyl-, acetate 875 0.14 ± 0.07

178

Ethyl pentanoate 898 0.19 ± 0.33

Myrcene 955 0.85 ± 1.09

Indene 1025 0.66 ± 0.14

Limonene 1031 0.54 ± 0.17

Trans linalool oxide 1088 11.69 ± 1.25 1.86 ± 0.1

Linalool 1098 1.19 ± 0.61

Myrcenol 1115 0.91 ± 0.12

2-Octen-1-ol acetate 1120 7.7 ± 1.17

Thujanol 1078 2.63 ± 0.56 0.54 ± 0.17

Citronellol 1178 0.4 ± 0.09

α-Terpineol 1185 2.44 ± 0.33

trans-Geraniol 1255 0.16 ± 0.21

Thymol 1285 0.1 ± 0.12

Limonene dioxide 1290 0.87 ± 0.11


Eugenol 1355 1.58 ± 6.03

Decanoic acid 1372 0.73 ± 0.02

Tetradecane 1415 0.68 ± 0.09

Pentadecane 1500 0.81 ± 0.05

Stearic acid 2075 1.74 ± 0.12


179

While only 10 aroma aglycones were detected in XAD, SPE isolate of drumstick was

characterized by the presence of 62 volatile compounds. The major aglycones identified

in XAD extract were 3-hexen-1-ol (2.5 µg/kg), benzene ethanol (5.94 µg/kg), benzyl

alcohol (4.35 µg/kg), 2-propyl heptanol (3.06 µg/kg), trans linalool oxide (11.69 µg/kg),

and 2-octen-1-ol acetate (7.7 µg/kg). Although the GC/MS profile obtained from

hydrolyzed XAD extract exhibited the presence of hydrocarbons and fatty acids such as

tetradecane, pentadecane, and stearic acid, they were not present in the SPE extract.

However, such compounds might not affect the overall aroma perception of the vegetable.

Alcohols constituted the most abundant chemical class in the SPE isolate, with 34 volatile

compounds. The isolate also contained 12 carbonyl compounds including 9 aldehydes and

3 ketones. The major alcohols and carbonyl compounds identified were 3-methyl-1-

butanol (12.11 µg/kg), 2-methyl-1-butanol (4.89 µg/kg), 3-hexen-1-ol (24.35 µg/kg),

hexanol (14.58 µg/kg), 2-ethyl-1-hexanol (9.32 µg/kg), 3-hydroxy-2-butanone (14.31

µg/kg), 3-methyl butanal (1.18 µg/kg), 2-hexenal (2.09 µg/kg) and 2-octanone (1.59

µg/kg). Most of these volatiles were also present in free form. Apart from these, several

terpenes along with fatty acids and esters were also detected in the bound aroma profile of

RTC drumstick. The major terpenes identified included limonene (0.54 µg/kg), linalool

oxide (1.86 µg/kg), linalool (1.19 µg/kg), α-terpineol (2.44 µg/kg), myrcene (0.85 µg/kg),

limonene dioxide (0.87 µg/kg), myrcenol (0.91 µg/kg) and eugenol (1.58 µg/kg). The

presence of glycosidic precursors of terpenes has been widely reported in literature [191].

However, to the best of our knowledge, no report exists on the nature of glycosidic aroma

precursors of drumstick pods.

180

Interestingly, unlike free aroma volatiles, very few nitrogen containing compounds were

detected in bound aroma profile of drumstick. As the hydrolysis was carried out with

pectinase, glucosinolates might not have been hydrolyzed to release nitriles or

isothiocyanates. However, the myrosinase enzyme present inherently in the vegetable

might have resulted in generation of the few such compounds (2-methyl butane nitrile,

isopropyl isothiocyanate and 1-propen-3-isothiocyanato) during processing.


Not all volatile compounds are responsible for the characteristic odor of a food product.

Consequently, there is a need to identify those that have a real olfactive impact on

drumstick. GC-O employing a sensory panel was used to determine the typical odor

notes of drumstick as described earlier (section 2.2.2.2.4.1). These odors were then

compared with volatile compounds identified by GC/MS in order to determine key

odorants of drumstick. Volatile aroma isolate was extracted by steam distillation using

simultaneous distillation extraction. The process was repeated several times and the

isolates obtained were pooled, concentrated and injected in GC/MS equipped with

Olfactometry Detection Port (ODP). The aroma volatiles isolated from HS-SPME were

also subjected to Global olfactometric analysis separately. Similar aroma notes were

perceived by the sensory panel in both the extracts. Table 32 represents different aroma

notes perceived by the sensory panel at different retention times. The compounds

responsible for the aroma note perceived was identified using MS at corresponding

retention times.

181

Table 32. ODP analysis of drumstick SDE oil

Rt (min) Aroma perceived KI Compound identified OAV

3.8 – 5.67 Buttery 812 Acetoin 0.03

7.75 Sulphur / rubber ND

8.2 – 8.7 Grassy green 815 hexanal 14.13

9.27 Sulphur / rotten 835 Isopropyl isothiocyanate ND

9.91-10.39 Flowery – green 857 Cis-3-Hexen-1-ol 0.404

10.7-10.9 B-complex / medicinal ND

12.11-12.39 Boiled potato ND

12.71- 13.13 Boiled drumstick/

cooked vegetable

946 Butane-1-isothiocyanato ND

13.45 Roasted peanuts ND

14.02-15.3 Burnt rubber/rubber 974 Isobutyl iso thiocyanate ND

18.23- 18.5 Flowery 1043 Benzene acetaldehyde 0.468

20.6-20.9 Smoky / beany 1093 Nonanal 3.44

22- 22.7 Roasted nuts 1198 Benzene isonitrile

23.5- 24 Boiled beany / boiled

seeds of drumstick

1204 2-Methoxy-3-isobutyl

pyrazine

2.5

24.7- 24.9 Boiled veg / fatty 1209 Decanal 210.5

26.00-26.30 Fresh cut drumstick 1215 Benzothiazole 0.24

27.08 – 27.2 Green drumstick 1261 2- Decenal 20.93

29.42 – 29.7 Fatty green 1291 2,4-decadienal (E,E) 31.42

32 – 32.8 Fruity/ leafy Damascenone ND

ND: Not Detected

182

Various aroma notes such as buttery, sulphury, grassy green, flowery, boiled drumstick,

drumstick seed, fatty/ beany, fresh-cut drumstick, fatty green were perceived by the

panelists. The nine judges constituting the sensory panel agreed that the odor of the

extract was typical drumstick like. A region corresponding to 26.19-29.4 closely

resembled to the odor of fresh-cut drumstick pods. The odor detection frequency

chromatogram of SDE volatile constituents analyzed by GC-O is shown in Figure 33.

Figure 33. Odor detection frequency chromatogram of drumstick SDE volatiles (Refer

table 32 for RI)

The highest detection frequency of 9 was observed for fresh-cut drumstick and green

drumstick aroma. Other odor notes readily perceived by the panel were grassy green,

flowery-green, boiled drumstick, roasted nuts and fatty green having a detection

frequency of 8. Many other odor notes such as sulphury/ rubbery, sulphury-rotten, boiled

potato, flowery, boiled beany, fruity/ leafy were perceived with comparatively lower

detection frequencies (Table 32). The identities of the compounds listed were established

0

1

2

3

4

5

6

7

8

9

10

812 815 835 857 946 974 1043 1093 1198 1204 1209 1215 1261 1291

DF

KI

183

based on their retention indices, odor quality, and mass spectral data with those of

standard compounds as listed in NIST and Wiley aroma compounds libraries. The

compounds identified corresponding to the aroma notes of fresh-cut and green drumstick

were decanal, benzothiazole and 2-decenal. Benzothiazole is reported to occur in various

fruits and vegetables such as potato [196], asparagus, mango and cocoa. It is reported to

possess sulfurous, vegetative, cooked, brown, nutty and coffee-like aroma. Alkanals and

alkenals are reported to contribute green-fatty/oily odors and are reported to be present in

several vegetables [135]. Unlike decanal and 2-decenal which possessed high odor

activity values, the OAV of benzothiazole was quite low, however, it was readily

perceived by the sensory panel.

To further ascertain the contribution of these compounds towards the characteristic aroma

of the vegetable, SDE oil of drumstick was loaded on a preparative TLC with hexane:

ether (80:20) as the developing solvent system. The TLC plate so developed was dried

under a slow stream of nitrogen until free of solvent and then subjected to sensory

analysis. The sensory panel sniffed the aromagram to locate the band corresponding to

drumstick aroma. Fresh-cut drumstick odor was perceived by most of the panel members

at a Rf value of 0.47. TLC plate when developed in iodine vapors resolved in six distinct

bands (Figure 34). However, the band corresponding to the characteristic drumstick

aroma could not be stained with iodine. Each band was separately scrapped and eluted

with double distilled diethyl ether. The ether was filtered and concentrated by a slow

stream of nitrogen. The concentrate so obtained was injected in GC/MS equipped with

ODP for characterization of the odor. The aroma notes were then perceived by the

184

sensory panel at the sniffing port. Table 33 represents the Rf values of various bands

observed along with the aroma perceived and major compounds identified.

Figure 34. TLC aromagram of drumstick SDE isolate.

The GC-O analysis of the band corresponding to drumstick aroma in TLC aromagram

showed the role of decanal, benzothiazole and 2-decenal as contributors to the

characteristic drumstick aroma. Thus the synergistic role of decanal, benzothiazole and 2-

decenal in contributing to the characteristic drumstick aroma was established. The

chemical structures and mass spectra of these compounds are shown in Figure 35.

185

Table 33. Bands isolated from TLC aromagram of drumstick oil with their odor and

major compounds identified.

Band Retention Factor

(Aroma perceived)

Major Compounds identified

Band 1

(bottom)

0 (seedy aroma) Diacetyl sulphide, S-(+)-1,2-

Propanediol, acetic anhydride,

palmitic acid

Band 2 0.125 ( fruity odor) 3-Hexen-1-ol, 1-Hexanol, Trans

linalool oxide, alpha-Terpineol

Band 3 0.26 ( Fruity / flowery) Linalool, alpha- terpinolene

Band 3a (region

between band 3

and 4; not visible

in iodine)

0.47 (fresh cut

drumstick aroma)

Decanal, Benzothiazole, 2- decenal,

Band 4 0.59 (Mild drumstick

note + sweet smell)

L-Linalool, nonanal, decanal, E-2-

Tetradecen-1-ol, Sulfurous acid, hexyl

octyl ester, Cis-9-Octadecen-1-ol

Band 5 0.72 ( no odor) Sulfurous acid, 2-ethylhexyl hexyl

ester

Band 6 (top) 0.91 (sulphurous smell) No compounds could be detected

186

Figure 35. Chemical structures and mass spectra of most potent odorants of drumstick

3.2.4.3. Pumpkin

3.2.4.3.1. Free aroma analysis

The GC/MS chromatograms of free aroma of pumpkin as obtained by SDE and SPME are

presented in Figure 36. Table 34 provides a quantitative distribution of the constituents

identified in the isolates obtained from both the techniques.

187

Figure 36. GC/MS profiles of SDE and SPME isolates of pumpkin

Table 34. Quantitative distribution (µg/kg of pumpkin) of volatile oil components

identified in SDE and SPME

Name KI SDE SPME

Alcohols

Ethanol <600 3.76 ± 0.08

1-penten-3-ol 685 0.84 ± 0.29

3-Methyl-1-butanol 735 1.67 ± 0.19

1-Butanol, 2-methyl- 742 0.61 ± 0.29

1-Pentanol 765 2.37 ± 0.06

Z-3-Hexenol 857 0.095 ± 0.02 0.88 ± 0.04

1-Hexanol 867 2.55 ± 0.29

188

(5Z)-Octa-1,5-dien-3-ol 983 1.34 ± 0.01

1-Octen-3-ol 985 19.29 ± 1.39

2 ethyl hexanol 1021 2.12 ± 0.03

benzyl alcohol 1035 0.53 ± 0.04

3-Octanol 1065 3.24 ± 0.25

2E,6Z-nonadien-1-ol 1159 1.23 ± 0.09 9.45 ± 1.65

2-Butyl-1-octanol 1250 0.19 ± 0.04

Dodecanol 1473 0.014 ± 0.05

1-Hexadecanol 1855 0.22 ± 0.12


Acetaldehyde <600 1.75 ± 0.34

2-Butenal 625 1.01 ± 0.39


1-Penten-3-one 680 0.69 ± 0.37

Pentanal 697 2.49 ± 0.49

(E)-3-Penten-2-one 725 0.15 ± 0.02

trans-2-Pentenal 745 0.59 ± 0.04

2-Methyl-3-pentanone 750 0.54 ± 0.01

Butanal, 2-ethyl-3-methyl- 781 0.36 ± 0.09


Hexanal 815 44.84 ± 5.62

2E-Hexenal 854 3.46 ± 0.76

189

Heptanal 899 0.71 ± 0.07

2,4-hexadienal 905 0.92 ± 0.14

(Z)-2-Heptenal 955 0.86 ± 0.02


3-octanone 981 0.41 ± 0.28

6-methyl-5-hepten-2-one 985 0.05 ± 0.01

2-Octanone 988 0.63 ± 0.24

2E,4E-heptadienal 995 1.78 ± 0.26

Acetophenone 1059 3.28 ± 0.58

(E)-2-Octenal 1060 1.26 ± 0.12

(Z)-6-Nonenal 1097 0.92 ± 0.14 2.48 ± 0.07

2E,4Z nonadienal 1154 0.68 ± 0.14

2E, 4E-nona-dienal 1213 0.106 ± 0.02 0.71 ± 0.01

Nonanal 1093 0.83 ± 0.03

Octadecanal 1357 0.13 ± 0.08

Dodecanal 1407 0.22 ± 0.01

Terpenes

Cis-limonene oxide 1085 0.09 ± 0.01

Linalool 1098 0.01 ± 0.004 0.2 ± 0.05

thujone 1117 0.36 ± 0.09

3-Caren-10-al 1125 0.009 ± 0.002

α-terpineol 1185 0.26 ± 0.04

190


Twenty one volatile aroma compounds were detected in the SDE isolate of pumpkin. It

consisted of alcohols (4), carbonyl compounds (3), terpenes (5) and nine other

compounds including nitrogen and sulphur containing compounds and fatty acids along

with their ester derivatives. The major aglycones present in the SDE isolate were Z-3-

Geranial 1197 0.39 ± 0.08

Trans-geraniol 1255 0.006 ± 0.001

β-ionone 1455 0.007 ± 0.002

Nerolidol 1550 0.019 ± 0.005

Others


2-Methyl pyridine 817 0.05 ± 0.01

Dimethyl sulfoxide 825 1.10 ± 0.05

1-Butanol, 3-methyl-, acetate 875 0.16 ± 0.05

Dimethyl sulfone 920 3.84 ± 0.92


Decanoic acid 1372 0.014 ± 0.01

Tetradecanoic acid 1768 0.078 ± 0.01

Hexadecanoic acid 1975 2.17 ± 0.16

9Z-Octadecenoic acid (oleic) 1949 1.025 ± 0.03

Octadecanoic acid 2075 0.578 ± 0.05

191

hexenol (0.095 µg/kg), dodecanol (0.014 µg/kg), 2E, 6Z-nonadien-1-ol (1.23 µg/kg),

hexadecanol (0.22 µg/kg), acetophenone (3.28 µg/kg), 6Z-nonenal (0.92 µg/kg), 2E, 4E-

nonadienal (0.106 µg/kg), dimethyl sulfone (3.84 µg/kg), hexadecanoic acid (2.17 µg/kg)

and 9Z-octadecenoic acid (1.025 µg/kg). Terpenes were detected in trace amounts in the

SDE oil.

SPME isolate on the other hand, was characterized by the presence of 48 volatile

compounds. It included 14 alcohols, 27 carbonyl compounds (20 aldehydes and 7

ketones), 5 terpenes and 2 esters. Aldehydes were the most abundant aroma compounds

in the SPME isolate. Hexanal (44.84 µg/kg) accounted for the highest content followed

by 3-methyl butanal (3.52 µg/kg), 2E-hexenal (3.46 µg/kg) and 6Z-nonenal (2.48 µg/kg).

Hexanal is known to contribute green notes in vegetables. The unsaturated C6 aldehydes

and particularly hexanal, has been postulated to be derived via lipid peroxidation during

processing and sampling [188]. Many of these aldehydes have a low human detection

threshold and are important for the flavour of tomato, cucumber, peppers and other

vegetables [197-199]. Among alcohols, major volatile compounds identified were 2-

methyl-1-propanol (1.52 µg/kg), 3-methyl-1-butanol (1.67 µg/kg), pentanol (2.37 µg/kg),

hexanol (2.55 µg/kg), 1-octen-3-ol (19.29 µg/kg), 2-ethyl hexanol (2.12 µg/kg), 3-octanol

(3.24 µg/kg) and 2E, 6Z-nonadien-1-ol (9.45 µg/kg).

Low boiling alcohols and aldehydes such as 1-penten-3-ol, 3-methyl butanol, pentanol, 3-

methyl butanal, 2-butenal, 1-penten-3-one, pentanal, and hexanal, which were present as

major aroma compounds in SPME isolate, could not be detected in isolate from SDE.

This could be due to the masking of the initial region of chromatogram obtained from

192

SDE isolate by the solvent employed. Further, the evaporation step during concentration

of the SDE isolate by nitrogen might have resulted in the loss of these highly volatile

aroma compounds. The SDE oil was also characterized by the presence of 2-methyl

pyridine, dimethyl sulfone and dimethyl sulfoxide, which were however, not detected in

the SPME isolate. Such compounds could have been derived from sugars and amino acids

via various chemical reactions such as Maillard reaction during SDE as a result of heat

treatment [200]. SPME gave significantly better qualitative and quantitative aroma profile

compared to that obtained from SDE and had a higher content of alcohols and carbonyls.

Higher extraction efficiency of SPME than SDE has been reported in previous studies by

various researchers [181].

The volatiles of fresh, cooked and canned pumpkin were studied earlier by Parliment et

al. [184]. Combined steam distillation/solvent extraction was employed for extracting the

volatiles of cooked and canned pumpkin while for fresh pumpkin, headspace

concentration was used by these workers. The individual aroma compounds were

identified by GC/MS. A total of 30 compounds were identified in the volatile extracts of

freshly cooked and canned pumpkins and one additional compound from the raw

vegetable. The major classes of compounds identified were aliphatic alcohol and carbonyl

compounds (15), furan derivatives (5), and sulphur containing compounds (3). The major

compounds reported to be present in the volatiles from the freshly cooked vegetable were

hexanol, (Z)-3-hexenol, 2-hexenal, hexanal, and 2,3-butanedione (diacetyl). However,

virtually all of the C6 aldehydes and alcohols were lost in the canned product with the

major components being 2-methyl butanal, pyridine, furfural, 2,3-butanedione, and 3-

193

methyl butanal [184]. Other interesting compounds identified in the freshly cooked

material were 3-(methylthio)propanal and a methylformylthiophene, and in the canned

product 2-methyltetrahydrofuran-3-one, 2,5-dimethylpyrazine, and dimethyltrisulfide. 2-

sec-butyl-3-methoxy pyrazine were shown to be present in trace concentrations (<2.5

ng/L juice) in raw pumpkin [184]. In the freshly cooked vegetable, hexanal (4.5 ng/L),

(E)-2-hexenal (17 µg/L), (Z)-3-hexenol (70 µg/L), and 2,3-butanedione (7 µg/L) probably

play important roles in its flavor. However, in the canned material, 2-methylbutanal (1.3

µg/L), 3-methyl butanal (2 µg/L), and dimethyl trisulfide (10 ng/L) were reported to be

the more important components. Apart from few C5-C6 aldehydes and alcohols, the

aroma profile obtained in the present study was found to be different from that earlier

reported. This difference might be due to different variety or geographical origin of the

vegetables studied.


No report exists on the nature of bound aroma glycosides of pumpkin till date. The

representative GC/MS chromatograms of the free aglycones obtained from their

precursors are shown in Figure 37. The quantitative distribution of these aglycones in the

two isolates is presented in Table 35.

194

Figure 37. GC/MS profile of hydrolyzed aroma glycosides of pumpkin obtained from

XAD and SPE isolates.

Table 35. Quantitative distribution (µg/kg of pumpkin) of volatile oil components

identified in XAD and SPE isolates.

Name KI XAD SPE

Alcohols

1-Penten-3-ol 685 0.76 ± 0.04

3-Methyl-1-butanol 735 9.67 ± 1.66

1-Butanol, 2-methyl- 742 5.52 ± 1.02

1-Pentanol 765 1.85 ± 0.65

2-Buten-1-ol, 3-methyl- 772 0.09 ± 0.03

Z-3-Hexenol 857 0.23 ± 0.04 1.08 ± 0.31

195

(E)-2-Hexen-1-ol 853 1.08 ± 0.38

1-Hexanol 867 0.131 ± 0.03 13.49 ± 2.18

Heptanol 965 0.34 ± 0.07

1 Octen-3-ol 985 0.195 ± 0.06 7.03 ± 1.31

Phenol 940 0.184 ± 0.05

3-Octanol 1065 0.558 ± 0.08 1.59 ± 0.48

2-Ethylhexanol 1021 0.519 ± 0.12 1.56 ± 0.23

Benzyl alcohol 1035 2.125 ± 0.28 1.02 ± 0.28

2-Octen-1-ol, (Z)- 1060 0.07 ± 0.01

1-Octanol 1065 0.37 ± 0.06

Phenylethyl Alcohol 1120 0.971 ± 0.04 0.64 ± 0.18

trans,cis-2,6-Nonadien-1-ol 1159 0.084 ± 0.01 0.66 ± 0.15

2,6-Dimethyl-octa-2,6-dien-1-ol 1235 0.193 ± 0.03

2-Methoxy-4-vinylphenol 1312 1.26 ± 0.14

Tetradecanol 1654 0.98 ± 0.19

Hexadecanol 1855 2.17 ± 0.33



1-Penten-3-one 680 0.22 ± 0.16

Hexanal 815 1.44 ± 0.31 1.61 ± 0.53

2-Hexenal 854 0.2 ± 0.08

2-Heptanone 870 0.13 ± 0.01

196

Heptanal 899 0.08 ± 0.02

Benzaldehyde 961 0.11 ± 0.02 0.2 ± 0.02


2-Octanone 988 1.59 ± 0.25

2-Hydroxy-benzaldehyde 1035 0.15 ± 0.01

Nonanal 1093 0.064 ± 0.02 0.18 ± 0.09

6Z-Nonenal 1097 2.15 ± 0.18 0.27 ± 0.07

Dodecanal 1407 0.06 ± 0.01 0.09 ± 0.03

3-Methoxyacetophenone 1415 8.78 ± 1.53

2Z-Decenal 1259 0.744 ± 0.24

Tetradecanal 1575 0.525 ± 0.13

Terpenes

Limonene 1032 0.021 ± 0.005 0.19 ± 0.09

Linalool 1098 0.41 ± 0.03

Methyl nonanoate 1225 0.25 ± 0.05

Isomenthol 1140 0.154 ± 0.06 0.68 ± 0.09

α-Terpineol 1185 1.03 ± 0.26

Cis-Limonene dioxide 1290 1.24 ± 0.6

E-Citral 1197 0.17 ± 0.03

cis-Geraniol 1255 0.33 ± 0.01

Vanillin 1412 0.894 ± 0.05

Others

197

Ethyl propionate 703 0.34 ± 0.04

Butyl acetate 815 0.095 ± 0.03

3-Methyl-1-butanol acetate 875 0.133 ± 0.02

Octanoic acid 1165 0.607 ± 0.09

2,3-Dihydro benzofuran 1225 0.844 ± 0.06

Nonanoic acid 1278 1.46 ± 0.15

Decanoic acid 1278 1.10 ± 0.13 0.32 ± 0.04

Menthyl acetate 1304 0.41 ± 0.24

Dodecanoic acid, methyl ester 1325 0.06 ± 0.001 0.54 ± 0.2

Dodecanoic acid 1565 13.96 ± 2.05

2-Octyl furan 1275 2.15 ± 0.24

Tetradecanoic acid, methyl ester 1695 0.15 ± 0.002 0.37 ± 0.05

Hexadecanoic acid, methyl ester 1935 3.04 ± 0.12

Eicosanoic acid, methyl ester 2105 0.28 ± 0.03

Octadecanoic acid 2075 3.89 ± 0.24

9Z-Octadecenoic acid 1949 8.37 ± 1.25


A total of 36 compounds were detected in the XAD extract of pumpkin. The major

chemical classes were alcohols (13), carbonyl compounds (7), terpenes (3), fatty acids

and esters (11). Two furan derivatives 2,3-dihydro benzofuran and 2-octyl furan were also

detected in the XAD extract. Although furan derivatives were reported earlier in

pumpkin by Parliment et al. they were not detected in the free form in the present study

198

[184]. This might be due to low concentration of these compounds which is beyond the

detectable limit of GC/MS. The major compounds identified could be classified as

alcohols: benzyl alcohol (2.12 µg/kg), phenyl ethyl alcohol (0.97 µg/kg), 2-methoxy-4-

vinyl phenol (1.26 µg/kg), tetradecanol (0.98 µg/kg), hexadecanol (2.17 µg/kg);

aldehydes: hexanal (1.44 µg/kg), 6Z-nonenal (2.15 µg/kg); fatty acids and esters:

nonanoic acid (1.46 µg/kg), decanoic acid (1.10 µg/kg), dodecanoic acid (13.96 µg/kg),

methyl ester of hexadecanoic acid (3.04 µg/kg), octadecanoic acid (3.89 µg/kg), 9Z-

octadecenoic acid (8.37 µg/kg); and 2-octyl furan (2.15 µg/kg).

The GC profile of the SPE isolate on the other hand, was characterized by the presence of

45 compounds with 17 alcohols, 14 carbonyl compounds, 8 terpenes and 6 fatty acids or

esters. Among alcohols, the major components were 2-methyl-1-propanol (8.55 µg/kg),

3-methyl-1-butanol (9.67 µg/kg), hexanol (13.49 µg/kg), 1-octen-3-ol (7.03 µg/kg), 3-

octanol (1.59 µg/kg) and 2-ethyl hexanol (1.56 µg/kg); while among carbonyl

compounds, 2-methyl butanal (1.33 µg/kg), hexanal (1.61 µg/kg), 2-octanone (1.59

µg/kg) and 3-methoxy acetophenone (8.78 µg/kg) were identified as the major

compounds. Major terpenes include α-terpineol (1.03 µg/kg) and cis-limonene dioxide

(1.24 µg/kg) with others present in comparatively lower amounts. Very few fatty acids

and esters were detected in the SPE isolate in comparison to that in the XAD extract.

Most of the alcohols identified in the glycosidic fraction were also detected in the free

aroma profile of the vegetable. Interestingly, many of the C5-C6 alcohols, carbonyl

compounds and terpenes, which were the major components in the SPE isolate were not

present in XAD isolate. Isolate from XAD was dominated by high boiling alcohols such

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as tetradecanol, hexadecanol, and fatty acids and esters, which were not present in the

SPE isolate. Owing to their low volatility, these compounds may not play a significant

role in contributing the characteristic aroma.


Many aroma compounds have been earlier reported in pumpkin. However, no sensory

correlation of the identified compounds with the actual odor of the vegetable was

established. It was therefore of interest to study the key odor compounds contributing

towards the characteristic aroma of the vegetable. GC-O analysis was separately carried

out using both SPME and SDE isolate. The headspace volatiles as extracted using SPME

fibre were desorbed (270 °C) in GC/MS equipped with ODP. The isolates obtained after

repeated simumtaneous distillation extractions were pooled, concentrated and also

subjected to GC-O analysis. Various aroma notes were perceived by a sensory panel of 9

members at the sniffing port. Table 36 represents various aroma notes perceived at

different retention times at the sniffing port along with the identities of the corresponding

compounds, established using MS data and RI values as reported in the literature. Figure

38 shows a plot between detection frequency and RI of the compounds identified

corresponding to the various aroma notes.

Table 36. ODP analysis of pumpkin aroma (SDE)

Rt (min) Aroma perceived Compound

identified

KI OAV

7.12-7.30 Grassy green hexanal 801 10

7.5-8.30 Sweet green ND

10.65-10.82 Fruity-green Heptanal 901 0.24

9.73-10.10 Fruity Hexyl acetate 1007 0.035

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11.05-11.29 Roasted peanuts/ popcorn Vinyl acetate 1015 ND

16.00-16.2 Husk (rice husk) 2-Ethyl hexanol 1021 7.85E-05

17.34-17.5 Mustard/ sulphury ND

18.00-18.4 Oily green Acetophenone 1059 0.05

21.6-21.76 Cucumber 2E, 6Z- Nonadienol 1159 9450

21.77-22.08 Pumpkin Z-6-Nonenal 1097 124

19.5-19.7 Fatty/oily Octadecanal 1357 ND

Figure 38. Odor detection frequency chromatogram of pumpkin aroma isolate (Refer

Table 36 for KI)

The highest detection frequency of 9 was observed for the odor notes of fresh cucumber

and pumpkin. Other aroma notes readily perceived by the sensory panel were grassy

green, roasted peanuts/popcorn type aroma with a detection frequency of 8. Few other

odors as perceived by the sensory panel with comparatively lower detection frequencies,

included fruity-green, husk, and oily notes. The characteristic pumpkin aroma was

perceived by all the panelists at a retention time 21.77-22.08 min. The corresponding

0

1

2

3

4

5

6

7

8

9

10

801 901 1007 1015 1021 1059 1097 1159 1357

DF

KI

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compound identified at this Rt was found to be Z-6-nonenal, previously reported as an

important aroma compound of melon. The odor note corresponding to pumpkin aroma

was preceded by cucucmber aroma which was found to be due to the presence of 2E, 6Z-

nonadienol. The aroma of 2E, 6Z- nonadienol has been described as cucumber like while

Z-6-nonenal is known to possess green cucumber and melon like with woody orris type

[201].

Figure 39. Chemical structures and mass spectra for most potent odorants of pumpkin

The unsaturated alcohols and aldehydes such as (E,Z)-2,6-nonadienol and Z-6-nonenal

are formed during oxidation of n-3 polyunsaturated fatty acid [202, 203]. (E,Z)-2,6-

nonadienol has been previously reported in muscadine grape juice with a relatively high

FD factor and in musts obtained from French and Romanian Hybrids of grapes [204,

205]. Higher odor activity values (OAV) of these two compounds (Table 36) further

suggested their significant contribution in the overall aroma of the vegetable. Thus, Z-6-

nonenal and 2E, 6Z- nonadienol could be proposed as the most potent aroma compounds

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of pumpkin. The chemical structures and mass spectra of these odorants are shown in

Figure 39.

3.2.5. Effect of radiation processing on free and bound aroma profiles of the selected

vegetables

Irradiation is recognized in many countries worldwide as a safe and effective method for

preservation of raw and processed foods. The main benefit of irradiation includes

eliminating microorganisms & insects or parasites capable of causing food spoilage and

toxicity. Fresh-cut vegetables are a growing class of food stuff that has received increased

interest and attention in recent years due to the convenience it provides to the consumer.

One of the most important problems of storage of fresh-cut produce is the maintenance of

taste and aroma that are essential parameters for their quality. Use of gamma irradiation

as a potential preservation technique for improving the shelf life of fresh-cut fruits and

vegetables due to the cold nature of the process holds promise.

Aroma composition plays an important role in the final quality of a food product.

Radiation processing has been reported to result in significant changes in volatile profile

of food products particularly those with high water content such as fresh produce [206].

Information on the impact of irradiation on the volatile composition in fresh produce is

limited. Hydrolysis of aroma precursors and a resultant increase in free aroma volatiles

during radiation processing has been recently reported. Radiation induced hydrolysis of

aroma glycosides has been successfully demonstrated in products such as saffron,

nutmeg, fenugreek and papaya [133, 207, 208, 209]. Reports on the effect of radiation

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processing on these constituents in Indian vegetables are scanty. To the best of our

knowledge no report exists on the effect of radiation processing on the free and

glycosidically bound aroma constituents of the currently selected vegetables. It was

therefore of interest to study the influence of radiation processing and storage on the

volatile aroma compounds and their precursors in the selected vegetables.

As evident from the GC/MS data obtained for all the three vegetables, SPME and SPE

techniques of extraction for free and bound aroma respectively were found to be better as

higher number of compounds were detected and the profiles were free of interference

from hydrocarbons or fatty acids as was the case with the other techniques employed.

Therefore the aroma profiles obtained by SPME (for free aroma) and SPE (for bound

aroma) for control and radiation processed samples at different storage durations were

further examined. The free and bound aroma compounds of ash gourd, drumstick and

pumpkin are depicted in tables 24-28 and 30-31. Apart from the compounds listed in the

tables, certain minor peaks, which are not identified here, might also undergo changes

during radiation treatment or storage. Therefore, data points at sufficiently small intervals

(every 0.17 min) in each chromatogram were taken into consideration while evaluating

the effect of processing steps. This however resulted in innumerable points which are

difficult to analyze manually. Therefore a data reduction technique, such as principal

component analysis (PCA) was employed, that can reduce the vast data points into a

fewer number. This aided in explaining the inherent variation of original data. It is an

unsupervised technique which is used for dimensionality reduction of multivariate data

sets and allows visualization of complicated data for easy interpretation [210].

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The GC/MS data obtained for free and bound aroma was therefore separately subjected to

principal component analysis (PCA) for each vegetable. To know the nature of the

constituents responsible for the differences among control and radiation processed

samples at different days, factor loading data obtained from PCA analysis for free as well

as bound aroma was analyzed separately for each vegetable.

3.2.5.1. Ash gourd

Principal component (PC) score plots obtained for free and bound aroma compounds of

ash gourd are represented in Figure 40 and 41, respectively. In case of free aroma, first

two principal components (F1 and F2) cumulatively explained 82.79 % of data variation

while in case of bound aroma, 87.74 % variation was accounted by F1and F2.

Figure 40. PCA of free aroma of ash gourd (C0, C5, C8, C12 – control samples of day 0,

5, 8 and 12; I0, I5, I8, I12 – irradiated (2 kGy) samples of day 0, 5, 8 and 12).

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Figure 41. PCA of bound aroma of ash gourd (C0, C5, C8, C12 – control samples of day

0, 5, 8 and 12; I0, I5, I8, I12 – irradiated (2 kGy) samples of day 0, 5, 8 and 12).

Analysis of GC spectral data of free aroma by Principal Component Analysis (PCA)

revealed changes in the volatile composition as a result of radiation processing (2 kGy)

and storage. The control samples at day zero were located in upper left quadrant (negative

side of F1 and positive side of F2), while all other samples got segregated from 0 d

control samples, and were grouped together. Careful interpretation of GC/MS data

revealed quantitative changes in the aroma compounds identified which could be

responsible for the segregation in the PC plots.

206

In case of alcohols, an increased content of 3-methyl-butanol, 2-methyl butanol, hexanol

and 2-ethyl hexanol was observed in radiation processed samples on day zero. An

increase of 81, 400, 298 and 66 % was observed in the contents of these alcohols

respectively as a result of radiation treatment (2 kGy). The glycosidic precursors of these

alcohols were also identified in the vegetable. A corresponding decrease in the respective

glyco-conjugates was observed. Thus, the increased content of free alcohols in radiation

processed samples could be the result of radiolytic breakdown of their glycosidic

precursors present in the vegetable. Further, the contents of these alcohols increased

during storage in control as well as radiation processed ash gourd. The increased content

of various alcohols during storage has also been reported earlier in cantaloupe and

watermelon [178]. They correlated increased content of alcohols with senescence of the

product. Aroma notes of 3-methyl-butanol, 2-methyl butanol, hexanol and 2-ethyl

hexanol are reported to be whisky, wine, sweet-green and citrus/ fresh floral respectively.

Aldehydes, known to impart “green” and “grassy” notes decreased as a result of radiation

processing as well as during storage. They are reported to play a relatively important role

in vegetable flavors [145]. A decrease of 45 % in the content of 3-methyl butanal, 66 % in

4-methyl-4-penten-2-one, 57 % in 2-pentanone, 35% in hexanal, 41 % in octanal and 62

% in decanal was observed. The C6-C8 aldehydes are known to be formed via the

lipoxygense pathway from unsaturated fatty acid precursors namely linoleic and linolenic

acids liberated mainly from galactolipids. Decreased content of aldehydes during storage

has also been reported earlier in fresh-cut cantaloupe [178]. Reduction of aldehydes to

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branched-chain alcohols, and their subsequent conversion to branched-chain acetates was

cited as the probable reason by these workers.

Among esters, no significant effect of radiation processing was observed except for ethyl

acetate. Ethyl acetate is known to possess fruity odor note, and is widely reported in

various fruits and vegetables [191]. Radiation treatment (1 kGy) resulted in 73 % decline

in the content of ethyl acetate on day zero as compared to the non-irradiated fresh control

samples. Decreased production of esters with increasing radiation dose (0.44 – 1.32 kGy)

was also reported earlier in apples stored at 20 °C [211].

A significant decrease in the contents of bound aroma precursors of terpenes was also

observed in irradiated samples during storage, indicating breakdown of their glycosidic

precursors due to radiation processing and during storage. A maximum decrease of 84 %

was observed for glycosidic precursor of sabinene, followed by β-pinene (83 %), indene

(79 %), limonene (41 %), and cymene (49 %) with a minimum decrease of 21 % in the

case of myrcene. Radiation induced breakdown of terpene glycosides has been widely

reported [133, 207-209]. Thus, the decrease in content could be due to radiation induced

hydrolysis of glycosidic precursors of these compounds. However, the degradation or

oxidation of free terpenes during extended storage could be the reason for the

insignificant change observed in their contents in free aroma. Generally terpenes

influence the aroma perception of a plant produce significantly. No major changes in the

terpene compounds during storage and radiation processing indicates that aroma quality

of the vegetable was not altered during the intended storage period (12 d).

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A decrease in content of aroma glycosides due to radiation processing has been reported

earlier in several food products such as coffee, nutmeg, fenugreek, and papaya [133, 207-

209]. In case of nutmeg, glycosidic precursors of p-cymene-7-ol, eugenol,

methoxyeugenol and α-terpineol were reported [133]. A radiation dose dependent

decrease in content of all glycosides was reported by these authors. In case of fenugreek,

radiation dose dependent decrease in content of phenol glycoside was reported [208]. In

coffee beans a decreased content of glycosidic precursors of isoeugenol and 4-

vinylguaiacol as a result of radiation processing was reported [212]. Thus, our results are

in accordance with already published literature data.

Among the key odorants, the content of acetoin increased in radiation treated ash gourd

on day zero (C- 12.16 µg/kg; I- 20.39 µg/kg). A decrease by 41 % in the content of

octanal was observed, while no significant effect of irradiation was noted in the nonanal

content. Although changes were produced by radiation treatment, none were sufficient to

be observed in a sensory test. Thus the overall sensory acceptability of the developed

product was unaltered with reference to aroma quality.

3.2.5.2. Drumstick

The PC plots for free and bound aroma of drumstick are presented in Figures 42 and 43,

respectively. The first two principal components (F1 and F2) explained 30.99 and 22.59

% variation in the data for free aroma and 55.32 and 15.10 % variation in the data for

bound aroma.

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Figure 42. PCA of free aroma of drumstick (C0, C5, C8, C12 – control samples of day 0,


PC plot for free aroma of drumstick shows that fresh control samples (C0) were

segregated from all the other samples (Figure 42). However, no clear segregation was

observed between the control and radiation processed samples at different storage days,

indicating no major changes in the free aroma composition of drumstick. In case of

bound aroma, only irradiated samples on day zero were grouped separately, while no

variation was observed among other stored samples (Figure 43).

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Figure 43. PCA of bound aroma of drumstick (C0, C5, C8, C12 – control samples of day

0, 5, 8 and 12; I0, I5, I8, I12 – irradiated (1 kGy) samples of day 0, 5, 8 and 12)

Careful interpretation of GC/MS data for free aroma compounds revealed some

quantitative differences among the compounds identified. Among the aldehydes, a two

fold increase in the content of hexanal and 58% increase in the content of trans-2-hexenal

were observed as a result of radiation processing. Hexanal is characterized by green,

grassy odor note, while trans-hex-2-enal possesses fresh green and leafy aroma. They are

reported to play a relatively important role in vegetable flavors [145]. Increased content

of these aldehydes due to radiation processing was previously reported in various

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vegetables. UV irradiation of tomato fruits and leaves was shown to increase the

production of n-hexanal as a result of enhanced lipooxygenase and hydroxyperoxidase

lyase activity [213, 214] also reported an increased trans-hex-2-enal content in soybeans

due to gamma irradiation at a dose above 10 kGy with as high as 5 times increase at 100

kGy. Fan and Sokorai, on the other hand observed an increase in trans-hex-2-enal content

of cilantro during post-harvest storage with no significant effect on the content of this

compound on irradiation [215]. An increase in the content of trans-2-hexenal was also

observed in irradiated cabbage [145]. Thus, increase in trans-2-hexenal might be

attributed to radiation induced lipid radiolysis resulting in release of linolenic acid and the

subsequent conversion of this fatty acid to this compound via lipooxygenase (LOX)

pathway to the aroma compound.

In case of terpenes, α-terpineol exhibited a two fold increase in response to radiation

treatment (1 kGy). α-Terpineol is a degradation product of limonene and linalool and rate

of formation from linalool is faster than from limonene [216]. A corresponding decrease

in content of linalool was also noted after radiation treatment on day zero (control: 2.02

µg/kg; irradiated: 1.13 µg/kg). Although the contents of linalool, limonene and α-

terpineol decreased during storage, the content of α-terpineol was higher in irradiated

samples than the corresponding controls at the end of intended storage period for 12 d

(control: 0.19 µg/kg; irradiated: 0.36 µg/kg).

Various nitrogen and sulphur containing compounds were also detected in the aroma

profile of drumstick. The major compounds were 2-methyl propane nitrile, 2-methyl

butane nitrile, isopropyl isothiocyanate, 2- butyl isothiocyanate, isobutyl isothiocyanate

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and 2-methyl benzonitrile. Many sulphur and nitrogen containing compounds such as

glucosinolates have been reported earlier in drumstick [113]. These glucosinolates

undergo hydrolysis in the presence of inherently present myrosinase enzyme in the

vegetable during storage or processing, giving rise to nitriles, isonitriles, thiocyanates and

isothiocyanates. An increase of 16 %, 79 % and 78 % in the content of isopropyl

isothiocyanate, 2-butyl isothiocyanate and isobutyl isothiocyanate was observed as a

result of radiation processing on day zero. Processing is often associated with increased

enzymatic activities, which could have resulted in the increased contents of these

components. However, no significant variation in the contents of other compounds was

noted in response to irradiation and storage. Although storage caused a decrease in the

contents of most of the compounds identified, the contents were significantly higher in

radiation processed samples as compared to control samples until the intended storage

period of 12 d.

Despite the changes in the contents of various aroma compounds as a result of radiation

processing and storage, no significant variation was observed in the contents of key

odorants of drumstick. Also, no change in aroma quality was perceived by the sensory

panel in irradiated drumstick samples.

Among bound aroma, appreciably more number of alcohols were identified which were,

however, not detected in the free aroma. It indicates that most of the alcohols are present

in the glycosidic form in drumstick. The major alcohols responsible for the segregation of

irradiated samples of day zero (I0) from others in bound aroma were 3-methyl butanol, 2-

penten-1-ol, 2-hexanol, 3-hexen-1-ol, hexanol, benzyl alcohol, and α-terpineol. The

213

content of these compounds in fresh control samples were 12.11 µg/kg, 1.16 µg/kg, 10.77

µg/kg, 24.35 µg/kg, 14.58 µg/kg, 7.34 µg/kg, and 2.44 µg/kg which increased to 38.86

µg/kg, 2.48 µg/kg, 18.92 µg/kg, 35.09 µg/kg, 94.18 µg/kg, 53.94 µg/kg, and 6.24 µg/kg

respectively in radiation processed samples. This significant increase could be due to

higher extractability of the aroma glycosides in radiation processed drumstick. Such

observations have been reported earlier in various food products [217]. Thus the results

obtained in present study are in agreement with the already published literature.

The contents of free terpenes released from their glycosidic precursors also increased

significantly in response to irradiation. An increase of 82, 300, 90, 82, 33, 131, 68 and 45

% was observed in the contents of citronellol (citrus with green fatty terpene nuances),

limonene (citrus), indene (fresh citrus grapefruit), linalool (sweet floral), α-terpineol (pine

floral), trans geraniol (floral), myrcene (spicy), and limonene dioxide (citrus)

respectively. Greater extractability of the aroma precursors in radiation treated samples

might have resulted in the higher amounts obtained. Terpenes generally have substantial

influence on the aroma of a product. The increased contents of these aglycones in

response to radiation treatment thus suggest improved aroma quality of the product.

3.2.5.3. Pumpkin

Score plots of both the aroma profiles are depicted in Figures 44 and 45, respectively. The

first two principal components (F1 and F2) explained 65.4 and 13.9 % variation in the

data of free aroma, while 96.4 and 3.1 % variation in the bound aroma was explained by

F1 and F2, respectively.

214

Figure 44. PCA of free aroma of pumpkin (C0, C7, C14, C21 – control samples of day 0,


Score plot of free aroma clearly indicates that there are no major changes in aroma profile

due to radiation processing or storage, except for the control samples stored for 21 d,

which segregated from the rest of samples. The contents of certain compounds such as

acetaldehyde (pungent), 2-methyl-1-propanol (musty), 3-methyl-1-butanol (whisky,

malt), 2-methyl butanol (wine, onion), ethanol (sweet) and 3-octanone (musty,

mushroom) were found to be higher in control RTC pumpkin after 21 days of storage as

compared to other samples. Most of these compounds are reported to be produced by

micro-organisms during extended storage due to spoilage [15]. In a study conducted by

[17], by inoculating mixed-lettuce agar with spoilage bacteria and yeast, various volatile

organic compounds such as ethanol, ethyl acetate, 2-methyl-1-propanol, 2-methyl-1-

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butanol, 3-methyl-1-butanol, 2,3-butanedione, 3-methyl-1-pentanol, 1-butanol and 1-

hexanol were identified in the head space as spoilage markers. Thus the role of these

compounds in contributing to the observed variation in volatile profile of pumpkin

samples could be explained.

Figure 45. PCA of bound aroma of pumpkin (C0, C7, C14, C21 – control samples of day

0, 7, 14 and 21; I0, I7, I14, I21 – irradiated (1 kGy) samples of day 0, 7, 14 and 21)

Among key odorants, the contents of 6Z-nonenal (C- 2.48 µg/kg; I-4.39 µg/kg) and 2E,

6Z-nonadienol (C-0.27 µg/kg; I-0.52 µg/kg) increased in radiation treated samples.

However, their contents were comparable to that of fresh control samples at the end of

216

storage period. Thus sensory panel could not distinguish between the aroma quality of

fresh control and irradiated samples stored for a period of 21 d.

Score plots (Figure 45) for bound aroma indicated that the radiation processed (1 kGy)

samples at zero day were different from rest of the samples. By careful interpretation of

GC/MS data, this variation was found to be mainly due to the decreased contents of

glycosidic precursors of pentanol (C-18.5 µg/kg; I-10.5 µg/kg), Z-3-hexenol (C-7.08

µg/kg; I- 3.63 µg/kg) and hexanol (C-23.49 µg/kg; I-12.06 µg/kg) on day zero. A

corresponding increase in the aglycone content was observed in free aroma profile. This

could be attributed to radiation induced hydrolysis of aroma glycosides, resulting in

increased aglycone content and decreased content of corresponding glycosides. Radiation

induced hydrolysis of glycosides has been widely reported in food products resulting in

enhanced aroma quality [218]. Thus an overall enhanced aroma quality was observed in

radiation processed RTC pumpkin.

3.2.6. Gamma irradiation induced browning inhibition

Studies so far have demonstrated the feasibility of using gamma irradiation for extending

shelf life of RTC ash gourd. Irradiated RTC ash gourd had a superior visual appeal

compared to the control samples as a consequence of surface browning inhibition (Figure

18). Development of browning in the control samples was also evident with a

significantly (p < 0.05) lower ‘L’ value (Figure 15 (a)). Among the physiological factors

limiting post harvest storage of fresh plant produce, enzymatic browning plays a major

role in reducing sensory quality and nutritional value of these products. Gamma

217

irradiation induced browning inhibition in cut vegetables has been previously reported by

some workers but reports on the mechanism of its inhibition in cut vegetables are scanty.

Browning in cut fruits and vegetables mainly involves metabolism of phenolic

compounds into their oxidized products [219]. In intact plants, phenolic compounds in

cell vacuoles are spatially apart from the oxidizing enzymes present in the cytoplasm.

Once tissues are damaged by cutting, grinding or pulping, the rapid mixing of the

enzymes and phenolic compounds as well as the easy oxygen diffusion to the inner

tissues results in a browning reaction. In response to tissue injury, phenylalanine

ammonia lyase (PAL) produces phenols which are then oxidized by polyphenol oxidase

(PPO) and peroxidase (POD) to o-quinones that further polymerize to brown pigments.

3.2.6.1. Evaluation of parameters responsible for browning

Control and radiation treated ash gourd samples were processed as detailed in section

2.2.1. Various parameters responsible for enzymatic browning such as electrolytic

leaching, changes in micro-structure, enzyme activities (PPO, PAL and POD), phenolic

composition and o-quinones were monitored at different storage intervals.

3.2.6.1.1. Electrolytic leaching

Electrolytic leaching signifies the electrolytes leaching out on the surface of cut

vegetables due to wounding while processing. The tissues also undergo softening due to

hydrolysis of cell wall components (pectin, cellulose and hemicelluloses) during stress

(including radiation treatment) and extended storage resulting in increased electrolytic

218

leaching. The extent of leaching was measured in terms of conductivity and

spectrophotometric absorbances at various wavelengths.

Figure 46. Effect of radiation treatment (2 kGy) and storage on (A) Conductivity (B)

Absrbance at 250, 280 and 320 nm (C) Browning index (absorbance at 420 nm) of RTC

ash gourd

a) Conductivity: The variation in conductivity of the vegetable extracts with radiation

processing during storage is shown in Figure 46 A. A significant increase in values of

conductivity was observed in radiation-treated samples on day zero. During storage, the

conductivity of control samples increased substantially, while it was unchanged in

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irradiated samples. This infers increased content of electrolytes leaching out on the

surface of control RTC ash gourd.

b) Absorbance: The absorbance measured at different wavelengths (250, 280 and 320

nm) in control and irradiated samples during storage are shown in Figure 46 B. A

significantly higher values of absorbance were obtained in control samples as compared

to irradiated during storage. It indicates greater extent of compounds leaching out of the

matrix. On the other hand, no change in the absorbance of irradiated samples during

storage indicates better integrity of the tissue structure during extended storage. The

browning index (absorbance at 420 nm) also increased significantly in control samples on

day 8 and beyond (Figure 46 C). The aborbance at 280 nm can be taken as a measure of

the phenolic constituents leaching out on the surface. Thus, increased absorbances at 280

and 420 nm indicate more leaching out of the phenolics on the surface of the vegetable,

which undergo aerial oxidation in the presence of PPO or POD enzymes, and

subsequently form brown pigments at the surface of the control samples. Increase in

browning intensity has also been reported earlier in minimally processed lettuce during

storage of 5 d [220]. Significantly lower leaching of electrolytes in case of irradiated ash

gourd could have resulted in better retention of the visual quality during the intended

storage period (12 d).

3.6.1.2. Changes in microstructure

Scanning electron microscopic (SEM) studies demonstrate significant variation in the

microstructure of control and radiation treated (2 kGy) samples at the end of storage

period (12 d) (Figure 47 A-C). In control samples, after a storage period of 12 d, the

220

surface microstructure was clearly changed, as indicated by collapsed and damaged

tissues ((Figure 47 C). On the other hand, the tissues of radiation processed samples were

observed to be undamaged (Figure 47 B). Thus SEM studies further support the findings

as observed by electrolytic leaching experiments.

Figure 47. Scanning electron micrographs of RTC ash gourd (A) Non-irradiated control 0

d, (B) Radiation treated (2 kGy, 12 d) and c) Non-irradiated control 12 d. Figures are with

500 x magnification.

3.2.6.1.3. Evaluation of enzyme activities

Alteration in phenolic metabolism is generally known to affect browning in cut

vegetables. PAL is the first enzyme in the phenylpropanoid pathway involved in synthesis

221

of phenolic compounds. Apart from PAL, PPO & POD are the other key enzymes

involved in browning development of fresh-cut produce. Figure 48 represents the

variation in activities of PAL and PPO with radiation processing and storage. No

significant change in the PAL activity was observed as a result of radiation processing on

day zero (Figure 48 A). During storage, the PAL activity increased continuously in

control samples. Several studies on cut lettuce have shown a wound induced

enhancement in PAL activity. Degl Innoceti [138], for instance, noted a significant

increase in PAL activity within 5 h, whereas Hisaminato et al [221] found maximum

increase after 3 days of storage. Murata et al [27] also found a significant increase in the

activity of this enzyme in lettuce after 3 days of storage that further increased on storage

up to day 6. Thus, the effect of wounding on PAL activity was found to vary with the

variety of lettuce. Stress induced enhancement in PAL activity has been extensively

reported in different plant tissues. Various stresses such as nutrient deficiencies, viral,

fungi, and insect attack are known to increase either PAL synthesis or activity in different

plants [219]. Wound induced enhancement in PAL activity has also been previously

reported in minimally processed potatoes [222] and cabbage [145]. In the present case,

microbial and physiological spoilage of ash gourd during storage might have induced a

stress which resulted in an increase in PAL activity. On the other hand, in radiation

processed samples, PAL activity remained unchanged during storage. Induction of PAL is

a defence mechanism of the living plant tissues from external stress. Therefore during

storage, as the radiation processed samples were protected from spoilage, the metabolic

activity of PAL enzyme remained subsided. Higher phenolic biosynthesis as reflected by

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increased absorbance at 280 and 320 nm in the control samples also support the pattern of

PAL activity observed.

PPO: PPO is a downstream enzyme in the phenylpropanoid pathway acting on phenols to

form o-quinone. Radiation induced decrease in PPO activity was observed immediately

after treatment (Figure 48 B). Although a gradual increase in the activity was observed

during storage in both the samples, the activity of the radiation treated samples was

significantly lower (p < 0.05) than corresponding control samples throughout the intended

storage period (12 d). Similar observations have been reported earlier in edible mushroom

(Pleurotus nebrodensis), where a significant (p < 0.05) radiation induced decrease in PPO

activity was observed at a dose of 1.2 kGy [223]. They also observed an increase in the

PPO activity in control as well as radiation processed samples during storage, although

the activity in controls was higher than the irradiated samples. Duan et al. also reported

lowering in PPO activity due to a probable change in the conformation of active sites on

exposure to radiation and a consequent inhibition of browning in Agaricus bisporus

[224]. The results obtained in the present study are thus in accordance with the existing

reports.

POD: POD is another enzyme ubiquitously present in plants, that in the presence of

hydrogen peroxide converts a number of phenolics to form o-quinone. However, its role

in enzymatic browning remains questionable mainly because of the low H2O2 content in

vegetable tissues [219]. Free radicals including H2O2 are generated due to water

radiolysis on irradiation. Thus analysis of POD activity is of significance in the present

study. POD activity was assayed in the presence of natural hydrogen donors (chlorogenic

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and caffeic acid). POD activities did not vary substantially during storage for both the

substrates (Table 37), thus ruling out its role in browning in RTC ash gourd.

Figure 48. Effect of radiation processing (2 kGy) and storage on (A) PAL activity and (B)

PPO activity

Table 37. Effect of radiation processing (2 kGy) and storage on POD activity of ash gourd

Day Control Irradiated (2 kGy)

POD1 POD2 POD1 POD2

0 7.25 ± 1.25a 5.65 ± 1.05

b 5.55 ± 0.55

a 4.85 ± 1.05

b

5 7.00 ± 0.95a 5.3 ± 1.25

b 6.25 ± 0.90

a 4.9 ± 0.99

b

8 6.05 ± 0.80a 4.95 ± 0.95

b 5.75 ± 1.10

a 5.2 ± 0.75

b

12 5.75 ± 0.70a 4.35 ± 0.55

b 5.1 ± 0.85

a 5.05 ± 0.55

b

Data are expressed as mean ± standard deviation (n = 9). Mean values in the same column

bearing same superscript shows no significant difference (p ≤ 0.05). POD activity is

represented in Δ A min-1

g-1

FW; POD1 = chlorogenic acid peroxidase activity and POD2

= caffeic acid peroxidase activity.

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3.2.6.1.4. Analysis of phenolic constituents

Phenolic compounds are the primary substrate for the enzymes involved in browning, and

also their nature and content plays crucial role in browning development of fresh-cut

produce. The nature of phenolic constituents and their quantitative distribution during

radiation processing and storage was therefore analyzed in RTC ash gourd. The major

phenolic constituents identified in ash gourd included phenolic acids (α-resorcylic, p-

hydroxy benzoic, chlorogenic, syringic, p-coumaric acid) along with catechin and

naringenin (Table 38). As development of browning started on day 8, phenolic contents

were compared only from day 8 onwards. The contents of p-hydroxy benzoic,

chlorogenic, caffeic, and p-coumaric acid were significantly higher (p < 0.05) in control

samples as compared to the corresponding irradiated products on day 8 and 12 (Table 38).

The increased content can be attributed to higher PAL activity as observed in control

samples during storage (Figure 48 A). These acids are highly prone to oxidation and give

rise to brown/black polymers during aerial oxidation in the presence of PPO or POD.

Thus, their higher content in the control samples could explain the browning of these

samples towards the end of the intended storage period.

No significant effect of radiation processing was observed on the contents of most of the

phenolic constituents in ash gourd, except chlorogenic and caffeic acid, which exhibited

significant decrease (p ≤ 0.05) (Table 38). Interestingly, a radiation induced enhancement

by 87 % in the content of α-resorcylic acid was observed on day zero. An enhanced

synthesis of phenolic acids such as benzoic and cinnamic acids as a result of increased

PAL activity under stress in plants has been established earlier [225]. The content of

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resorcylic acid was significantly higher in irradiated samples as compared to controls on

each day of storage (Table 38). The acid is known to be a potential inhibitor of PPO.

Billaud et al. have reported an inhibition in browning of gum Arabic as a result of

inhibitory activity of α-resorcylic acid towards PPO isolated from this exudates [226].

The increased resorcylic acid content in irradiated RTC ash gourd could thus possibly

explain the prevention of browning in the treated product.

Table 38. Quantitative distribution of different phenolic constituents in ash gourd

Data are expressed as mean ± standard deviation (n = 6). Mean values in the same row

bearing same superscript shows no significant difference (p ≤ 0.05).

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3.2.6.1.5. Correlations between various factors contributing in browning inhibition

Correlation coefficients were calculated between various attributes involved in browning

phenomenon (Table 39). A high correlation between browning index (BI) and enzyme

activities (PAL and PPO) was noted in the control samples, indicating their significant

roles in browning. A positive correlation between browning and PPO activity has also

been reported earlier in peaches [227]. A moderate to high correlations were also obtained

between BI and contents of various phenolic acids (caffeic, chlorogenic and p-coumaric

acid). Thus an enhanced content of these acids could result in browning during storage.

Further, a negative correlation between BI and α-resorcylic acid content and between the

PPO activity and α-resorcylic acid provides evidence for the decreased PPO activity in

irradiated samples as a consequence of the increased content of this phenolic acid.

Therefore, its role in PPO inhibition and subsequently in browning inhibition during

storage was further investigated.

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Table 39. Correlation coefficients calculated between various factors contributing in

browning of RTC ash gourd

3.2.6.1.6. Role of α-resorcylic acid in inhibition of ash gourd PPO - Kinetics studies

A resorcylic acid concentration dependent decrease in browning was observed when

catechol was used as substrate (Figure 49 A). This observation demonstrated the role of

α-resorcylic acid in browning inhibition. The nature of inhibition and kinetic parameters

of enzyme inhibition were therefore further investigated.

The type of inhibition was deduced from Lineweaver-Burk (LB) double-reciprocal plot

(Figure 49 B). The linear plots obtained between 1/S and 1/V at different concentrations

of resorcylic acid were found to intersect on the left of the vertical axis and above the

horizontal axis. A decrease in Vm and an increase in Km was thus noted with increasing

concentrations of resorcylic acid. Thus inhibitory mode of resorcylic acid was found to be

of mixed type as both- slope as well intercept of the LB plot changed with different

228

concentrations of the inhibitor. This type of inhibition indicates that resorcylic acid

affected the affinity of the enzyme for catechol but did not bind at the active site of the

enzyme.

Figure 49. (A) Effect of resorcylic acid (0, 50, 125 and 250 mM) on the browning

reaction rate of catechol. (B) Lineweaver-Burk plot of resorcylic acid inhibition on the

catechol-ash gourd PPO system

The Ki (rate constant for reaction between enzyme (E) & substrate (S)) and Kis (rate

constant for reaction between enzyme-sbustrate complex (ES) & inhibitor (I)) values

were determined from the graphs between 1/Vmax vs [I] and Km/Vmax vs [I]

respectively (Figure 50 A & B). The value of Ki << Kis , implied a competitive

inhibition, with higher affinity of inhibitor for free enzyme rather than ES complex. In

inhibition studies of α-resorcylic acid on PPO from gum Arabic, a quasi-noncompetitive

inhibition with Ki value around 5 times lower than Kis value was noted [228]. Inhibitory

effect by various phenolic acids on PPO from various sources have been reported [228,

229]. PPO from different sources has been shown shown to be inhibited by phenolic

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compounds via different mechanisms. To the best of our knowledge, this is the first report

on inhibition of ash gourd PPO by α-resorcylic acid.

Figure 50. Plots for determining various kinetic parameters. (A) Plot of 1/Vmax vs [I];

(B) Plot of Km/Vmax vs [I]; (C) Plot of V0/(V0-Vi) vs 1/[I] (where V0 is the initial

velocity without inhibitor and Vi is the initial velocity in the presence of inhibitor); (D)

Inhibitory effect of different concentrations of α-resorcylic acid on the oxidation of

catechol by ash gourd PPO.

The reversible nature of inhibition of ash gourd PPO by α-resorcylic acid using catechol

as substrate was demonstrated from a plot of V0/(Vo-Vi) vs 1/[I] (Figure 50 C; V0 –

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velocity of reaction in absence of inhibitor; Vi – velocity of reaction in presence of

inhibitor). An intercept value > 1 on Y-axis implied reversible nature of inhibition. The

IC50 value was determined by a calibration graph between percent inhibition and [I]

(Figure 50 D). At a concentration of 25 mM, α-resorcylic acid exhibited 50 % inhibition

in the ash gourd PPO activity.

Thus the role of α-resorcylic acid in PPO inhibition was demonstrated. The nature of

inhibition was found to be mixed type, competitive and reversible, with IC50 value of 25

mM. Hence, radiation induced enhancement in the content of resorcylic acid could

contribute towards PPO inhibition, and thus preventing browning in radiation processed

RTC ash gourd.

3.3. Isolation, identification of bioactive constituents and determining their changes

during radiation treatment

There is an increasing interest in identifying bioactive components in native plant foods

and determining their role in contributing to the functional and nutraceutical properties of

these foods. This has resulted in concerted efforts towards identifying such foods

possessing novel bioactive organic molecules. Indian vegetables have traditionally been

used as medicinal ingredients in Ayurveda due to their unique bioactivities. Despite being

a treasure house of active constituents, very few studies exist on the nature of the

constituents present in majority of the Indian vegetables.

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3.3.1. Ash gourd

Ash gourd (Benincasa hispida) is known for its medicinal values and healing power and

has been widely used as a vegetable as well as a traditional medicine in the orient. As a

rich source of functionally important bioactives and therapeutics such as triterpenes,

phenolics, sterols and glycosides, the fruit has been widely used for the treatment of

epilepsy, ulcer, and other nervous disorders in the traditional medicinal systems of Asia.

The juice of the vegetable is recommended to patients suffering from heart ailments and

high blood pressure. Ash gourd waste (peel and seeds) has been traditionally used as a

green manure for increasing the soil nutrients while shelled seeds are reported to have

anabolic properties that promote tissue growth. The vegetable extract was therefore

screened for the bioactive principles responsible for some of the above activities.

3.3.1.1. Plant growth promoting activity

Restriction on the application of chemicals to enhance plant growth due to their negative

effects has resulted in increased interest in the isolation of novel natural plant growth

promoters. This has led to the identification of several newer plant growth regulators such

as jasmonates, brassinosteroids, salicylic acid, plant peptide hormones, polyamines, nitric

oxide, strigolactones and karrikins that influence various physiological processes such as

rooting, flowering, senescence, organogenesis or growth. In recent years, airborne

volatiles identified as acetoin and 2,3-butanediol from growth promoting strains of

Bacillus subtilis and Bacillus amyloliquefaciens have also been shown to stimulate

growth of Arabidopsis thaliana [230-232]. Microbial and plant derived compounds with

232

bio-regulatory activity thus represent a large pool of chemicals that show promise for

development of novel agrochemical compounds. Acetoin was found to be a major aroma

compound existing both in the free as well as in the bound form of the vegetable. As this

compound has been earlier shown to have growth promoting activity, it was of interest to

understand its role in the known growth promoting activity of the vegetable.

3.3.1.1.1. Isolation and identification of plant growth regulating compound

The total aqueous extract of ash gourd was fractionated into non-polar, medium polar and

polar fractions by using n-hexane, ethyl acetate and n-butanol, respectively. Except n-

butanol extract, all other fractions including the remaining aqueous residue had no growth

promoting activity. The butanol fraction was therefore used for subsequent isolation of

the active principles. Presence of activity in the n-butanol fraction suggested a polar

nature of the active compound(s). When subjected to TLC, the butanol fraction resolved

in five distinct bands (Rf values of 0.38, 0.44, 0.52, 0.59 and 0.77), while no clear

separation could be observed in the total aqueous extract. The component(s) present in the

major band at Rf 0.44 alone exhibited growth promoting activity. Acid hydrolysis of the

methanol eluate of this band and subsequent GC/MS analysis of the ether extract of the

hydrolyzate resulted in the identification of acetoin as its sole constituent. Analysis of the

acetylated derivative of the hydrolyzate remaining after diethyl ether extraction indicated

the presence of glucose thus suggesting the major active constituent to be acetoin

glucoside. The structure of the compound (TLC band, Rf 0.44) was further confirmed by

IR and NMR spectral analysis (Figure 51). The absorption band at 1665 cm-1

in IR

spectrum indicated the presence of carbonyl group in the molecule. The NMR signals at δ

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2.01 and 4.22 confirmed the presence of keto methyl and oxy methine group while a

doublet at δ 1.30 was assigned for the terminal methyl group in the acetoin moiety. A

broad signal in the range of δ 4.5-5.2 corresponding to sugar protons confirmed the

presence of carbohydrate moiety in the molecule. The IR absorption band at 910 cm-1

and

NMR peak at δ 4.2 (anomeric proton) suggested β- nature of the glycosidic linkage. The

β-linkage of the glucosidic bond was further confirmed based on the ability of

commercially available β-glucosidase enzyme to hydrolyze purified natural acetoin

glucoside. The chemical structure of the acetoin glucoside was further substantiated by its

chemical synthesis. Thus the structure of the molecule was identified as acetoin-β-

glucopyranoside.

3.3.1.1.2. Growth promotion studies

The effect of in vitro treatment of total aqueous extract of ash gourd, acetoin glucoside

(TLC isolated band) and standard acetoin at different concentrations (0.178 – 4.4 µg/mL)

on the diameter of tobacco leaf discs on day 14 is presented in Figure 52A. A significant

increase (p ≤ 0.05) in the leaf disc diameter was observed in all the treatments compared

to the control. Highest growth promotion activity was noted with acetoin glucoside

followed by acetoin and aqueous extract. An increase in diameter was observed up to a

concentration of 0.44 µg/mL in all the treatments. Beyond this concentration the

diameter was higher than the control, but lower than that observed at concentrations of

0.44 µg/mL in all the cases. Thus, an optimum concentration of 0.44 µg/mL was inferred.

A toxic effect on the leaves could possibly explain the retardation in growth of the leaf

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discs beyond this concentration. At a concentration of 4.4 µg/mL, complete inhibition in

growth was observed for all the treatments.

Figure 51. IR and NMR spectra of acetoin-3-O-β-D glucoside

A

B

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Figure 52. A) Effect of varying concentration (0.178 – 4.4 µg/mL) of total aqueous

extract (Aq extract) of ash gourd, acetoin glucoside and acetoin on tobacco leaf disc

diameter on day 14 (Different letters indicate significant differences) B) Effect of the

various treatments at the optimum concentration (0.44 µg/mL) on the diameter of leaf

discs during different time intervals (Different letters indicate significant differences).

236

Figure 52B shows the effect of various treatments at the optimum concentration

(0.44 µg/mL) on the diameter of leaf discs during different time intervals. A significant

increase (p ≤ 0.05) in diameter by 44 % (0.9 cm), 56 % (1.13cm) and 50 % (1.0 cm) was

noted in leaf discs treated with acetoin, acetoin glucoside and total aqueous extract

respectively on 14 d. In contrast, diameter of control leaf discs remained almost constant

during the entire time period studied. Beyond 14th

day, the leaf discs underwent decay and

hence the experiments were not continued further. Acetoin glucoside was found to induce

maximum growth response. This could possibly be due to the ready uptake of the

glucoside in comparison to its aglycone by the leaf discs. Figure 53A depicts the

enhancement in leaf disc diameter by acetoin glucoside at the optimum concentration of

0.44µg/mL on day 14. In a study on the growth promoting activity of volatile compounds

produced by bacterial strains GB03 and IN937a, Ryu et al [231] found that concentration

of acetoin released by these bacteria over a period of 24 h were 12 ± 5 µg and 8.8 ± 2.2

µg respectively, while concentration of 2, 3-butanediol was reported to be 3.9 ± 0.7 and

1.9 ± 0.5 µg, respectively. Induction of growth at lower concentrations in the present

study could be due to the greater uptake of these compounds by the leaf discs during

soaking unlike earlier studies on PGPRs, where restricted diffusion of volatiles into the

leaf from the atmosphere reduces this effect. The present study thus demonstrates that

acetoin and its glucoside possesses growth promoting activity.

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Figure 53. A) Increased leaf disc diameter for acetoin glucoside (0.44 µg/mL) treatment

on day 14. B) Germination in pearl millet seeds after treatment with acetoin and its

glucoside

In order to further confirm the plant growth promoting activity of acetoin and its

glucoside, pearl millet seeds were soaked in acetoin and acetoin glucoside (0.44 µg/mL

each) overnight and then sowed in pots (5 seeds per pot) containing autoclaved soil and

cocopet (1:1). Seeds soaked overnight in sterile distilled water were used as control. The

treatment by acetoin glucoside enhanced leaf length (33.5 ± 3.3 cm) and shoot length (5.5

±1.5 cm) when the seeds were allowed to germinate and grow for a period of 15 days

(Figure 53B). In contrast, the control and acetoin treated samples had similar leaf and

shoot length (25.5 ± 2.6 cm and 2.8 ± 0.8 cm respectively). High water solubility of

acetoin glucoside unlike acetoin could explain the observed enhancement in plant growth

when treated with the former. It is likely that once absorbed into the seed, the glycoside

could be hydrolyzed to acetoin that then promotes germination and growth. This however

needs further confirmation.

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3.3.1.1.3. Identification of differentially expressed protein in acetoin glucoside

treated tobacco leaf discs

Figure 54. Two dimensional gel from protein preparations of control and acetoin

glucoside treated tobacco leaves; Xt- protein over expressed in treated sample, Xc –

corresponding protein in control sample.

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Figure 55. Mass spectrum obtained by MALDI-ToF analysis of the protein (Xt) over-

expressed in response to acetoin glucoside treatment.

Figure 54 depicts the 2-D gel electrophoresis images of control and acetoin glucoside

treated tobacco leaves. In the present study we report the identification and bioinformatic

characterization of a protein (Xt) expressed in response to acetoin glucoside treatment.

Analysis of this protein by MALDI-TOF provided information on the peptide molecular

mass. The mass spectrum of this protein is shown in Figure 55. Searching the MASCOT

database yielded the best hit as nuclear GTP binding protein RanB1 in tobacco (Nicotiana

tabacum; Swiss Prot accession No. P41919). The best fit peptide masses in tobacco Ran

B1 are shown in Figure 55. Ran is a small nuclear protein (25 kD) with multiple

240

regulatory roles [233]. Evolutionarily conserved Ran belongs to Ras superfamily and has

been demonstrated to be a very important nuclear transport regulator [234]. Ran toggles

between GTP and GDP bound forms. A GTP bound form is activated for its GTPase

function by RanGAP protein while a new GTP molecule replaces the bound GDP by the

action of RanGEF [235]. There are local concentration variation of these two Ran

modifying proteins within the cell causing concentration gradient of RanGTP and

RanGDP [236]. This concentration gradient is believed to be important for the regulatory

functions of Ran in nuclear transport. It also plays a very important role in DNA

replication and cell cycle progression [237]. It regulates the spindle assembly before

chromosome separation, and also the nuclear envelop formation after mitotic

chromosome separation is completed. Overexpressed Ran1 in transgenic Arabidopsis and

wheat showed increase in cell number [238]. Transgenic arabidopsis showed distinct

phenotype such as increased tiller number, weak apical dominance, excess rosette leaves,

and wider siliques. Therefore, from studies carried out by these researchers, it is evident

that Ran is an important protein for cell division and tissue growth. As observed in our

experiments, acetoin and its glucoside from ashgourd increased the biomass production.

We find the presence of RanB1 among the upregulated proteins quite significant.

However it is unclear whether acetoin and/or its metabolic derivative(s) directly work on

Ran up-regulation or other regulatory proteins are also involved. Ran and its regulatory

functions are at the cross roads of many biochemical pathways. Observation of Ran up-

regulation in response to ashgourd derived acetoin glucoside treatment sheds a new light

on the mechanism of acetoin mediated growth enhancement. Currently our efforts are

241

concentrated on identifying the other overexpressed proteins in response to treatment of

plant tissue with acetoin glucoside. This will provide a greater insight into the mechanism

of acetoin action. The treatment of plants with highly active but cheap chemicals such as

acetoin and acetoin glucoside could have potential use in increasing crop yield.

3.3.1.2. Angiotensin converting enzyme (ACE) inhibition activity

Ash gourd has been used in traditional Indian and Chinese medicine to treat inflammation

and high blood pressure. Its juice is recommended to patients suffering from heart

ailments and high blood pressure. Inhibition of Angiotensin I Converting Enzyme (ACE,

kinase II, EC 3.4.15.1) is currently considered to be a useful therapeutic approach in the

treatment of high blood pressure. It is of great importance for controlling blood pressure

by virtue of the rennin-angiotensin system. This enzyme cleaves the C-terminal of

histidyl-leucine of the inactive decapeptide angiotensin I to form the octapeptide

angiotensin II, a potent vaso-constrictor [239]. Although synthetic ACE inhibitors such

as captopril are remarkably effective, they cause adverse side effects. Therefore, research

to find safer and naturally occurring ACE inhibitors is desirable for the prevention and

remedy of hypertension. ACE inhibitory peptides have been isolated from various foods

as well as from enzymatic digestion of food proteins such as buckwheat, chickpea, and

mushroom [240-242]. The ash gourd juice was therefore screened for ACE inhibition

activity.

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3.3.1.2.1. Activity guided fractionation

The ash gourd juice was fractionated with solvents of different polarity (hexane, ethyl

acetate and water) and the individual fraction was screened for ACE inhibitory activity by

an enzymatic assay using hippuryl-histidine-leucyl (HHL) as substrate (section 2.2.3.2).

The activity was mainly observed in the aq. extract left after partitioning with hexane and

ethyl acetate, while organic layers were devoid of ACE inhibitory activity. The aq extract

obtained from non-irradiated control and irradiated ash gourd at a concentration of 50

mg/mL exhibited 47.28 % and 40.50 % ACE inhibition, respectively. Thus, change in the

activity as a result of radiation treatment was insignificant. An ACE inhibition by 64 %

was reported earlier by ash gourd pulp extract at a concentration of 10 mg/mL [243].

They demonstrated that the inhibition of ACE activity by ash gourd fruit may be due to its

high phenolic contents. However, no compounds were identified by these researchers

responsible for this inhibition. The lower inhibition activity observed in the present study

could be due to the difference in variety and geographical origin of the vegetable studied.

The potency of an ACE inhibitor is usually expressed as IC50, which is equivalent to the

concentration of the compound inhibiting 50 % of ACE activity [244]. The IC50 value

was thus determined by regression analysis of ACE inhibition (%) versus different

concentrations of the extract. IC50 values for control and radiation processed ash gourd

extracts were found to be 3.77 and 4.4 mg/mL, respectively. The aq. extract was further

subjected to chromatographic separations for isolation and identification of the active

principles.

243

3.3.1.2.2. Isolation and identification of active principles

The aqueous extract obtained after partitioning with organic solvents from non-irradiated

control ash gourd was fractionated by HPLC using C18 column. Figure 56 shows HPLC

chromatogram of the total aq extract of ash gourd. For preliminary screening, different

regions of the HPLC chromatogram were collected and assayed for ACE inhibitory

activity. As evident from table 40, maximum activity (86 %) was exhibited by fraction 1

(F1, 2 – 4 min), followed by F2 (41 %), F3 (36 %) and F4 (20 %). All others fractions

collected were devoid of ACE inhibitory activity. The eluate collected corresponding to

F1 was therefore subjected to re-chromatography. After re-chromatography, four major

peaks (F1-1, F1-2, F1-3 and F1-4; Table 41) were collected separately to identify the

active compound. The activity mainly resided in the compounds eluting between 2.75-

3.00 min (F1-2). A positive reaction of this fraction with ninhydrin (development of

pink/purple color) indicated its peptide nature.

Figure 56. RP-HPLC profile of ash gourd aq extract

244

Table 40. The peaks collected from HPLC for preliminary screening of the fractions

possessing ACE inhibition activity.

245

Table 41. Fractions collected from re-chromatography of region 2-4 min obtained from

preliminary HPLC analysis.

The active fraction (F1-2) collected from repeated HPLC injections was pooled,

evaporated, and made up in aqueous solution (10 %). The aq solution so obtained, was

subsequently subjected to gel permeation chromatography (GPC), which gave rise to

three major peaks (Figure 57). Each of these peaks were collected and tested for their

ACE inhibitory activity. The activity mainly resided in fraction F1-2b (47 %) at a

retention time of 38.9 min (Table 42).

Figure 57. Chromatogram obtained from GPC of fraction 2

246

Table 42. Fractions collected from GPC of F1-2

For determining the molecular weight of active fraction (F1-2b), standards of known

molecular weight such as Leucine enkephaline (555.6 Da), Glu-Val-Phe (393.4 Da) and

Asp-Phe (280.28 Da) were injected in GPC. The molecular weight of the active fraction

(323 Da) was then calculated from the calibration curve plotted between molecular

weight and V/V0 (V - elution volume of the compound; V0 - void volume of the column).

The mass data indicated that peptide could be comprised of 2-4 amino acids. The purified

fraction showed a UV absorbance at 220 and 254 nm. Absorption at 254 nm indicates

presence of an aromatic amino acid in the active peptide. The active fraction (F1-2b) was

subjected to mass spectral analysis using direct probe facility available in GC/MS

(Shimadzu Corporation, Kyoto, Japan). Mass spectral data showed the presence of amino

acids viz. alanine (M+, 89) and valine (M

+, 117) in this fraction. The mass spectra of these

compounds are shown in Figure 58. However, the third possible amino acid present in the

peptide could not be determined in mass spectral studies. Many earlier studies on ACE

inhibitory peptides including from mushroom and broccoli have reported their nature to

be tripeptide [242, 245]. Cheung et al., reported the results of a series of inhibitory

peptides against ACE, indicating that aromatic amino acids at the C-terminal and

branched chain aliphatic amino acids at the N-terminal were suitable for a peptide binding

247

to ACE as a competitive inhibitor [246]. However, other reports have demonstrated that

inhibitory peptides possess an aliphatic amino acid residue at their C-terminal. The results

obtained in the present study, thus are consistent with the earlier reports. The

confirmation of the exact sequence of the amino acids in the active peptide, however,

needs further investigation.

Figure 58. Mass spectra of the identified amino acids viz. alanine and valine in the active

fraction

Thus, the consumption of ash gourd may provide significant protection against high blood

pressure, and inflammation regulated by rennin-angiotensin system. This study provides

evidence for pharmacological use of ash gourd in treatment of high blood pressure in

traditional Indian medicine. To the best of our knowledge, this is the first report on effect

of radiation processing on the ACE inhibitory activity of ash gourd. Therefore, radiation

processing could be employed for improvement in the shelf life of RTC ash gourd

without any substantial change in its ACE inhibition activity.

248

3.3.2. Cucurbitacins in pumpkin

Pumpkin belongs to Cucurbitaceae family and is known to possess several

pharmacological properties such as antidiabetic, antihypertensive, antitumor, immune

modulation, antibacterial, anti hypercholesterolemia, intestinal antiparasitia, and anti-

inflammation [247]. The ethanolic extract of the vegetable (Cucurbita pepo cv dayangua)

has been reported to show dose-dependent inhibitory effect on the growth of tumor cells.

This anti-tumor activity, has been attributed to the presence of various cucurbitane and

hexanorcucurbitane glycosides and other types of triterpenoids [248, 249]. Cucurbitane

glycosides such as cucurbitacin L 2-O-β-D-glucopyranoside, cucurbitacin K 2-O-β-D-

glucopyranoside; hexanorcucurbitane glycosides: 2,16-dihydroxy-22,23,24,25,26,27-

hexanorcucurbit-5-en-11,20-dione 2-O-β-D-glucopyranoside and 16-hydroxy-

22,23,24,25,26,27-hexanorcucurbit-5-en-11,20-dione 3-O-α-L-rhamnopyranosyl-(1 -->

2)-β-D-glucopyranoside have been isolated and identified in cucurbita pepo cv dayangua

[250]. Presence of cucurbitane triterpenoids with a purine unit and cucurbitacin C & E

has also been documented in pumpkin [248, 251]. Being associated with various

pharmacological activities and wide occurrence, it was of interest to explore the

occurrence of these bioactive constituents in pumpkin in the present study and determine

the effect of radiation processing thereon.

3.3.2.1. TLC analysis

The 1% methanolic extract (as obtained in section 2.2.3.5.) of control and radiation

processed samples stored for different storage periods (0, 7, 14 and 21 d) were subjected

to TLC analysis. Two major bands at Rf values 0.21 and 0.32 (Figure 59) and a minor

249

band at Rf 0.6 was observed when the plate was stained with iodine vapors. Low Rf

values of the two major bands suggested a polar nature of these compounds. In order to

identify the nature of the compounds in these bands, the plate was sprayed with

Liebermann-Burchard reagent (5ml acetic anhydride + 5ml conc. H2SO4 + 50 mL

ethanol). Development of pink colored spots when the plate was heated at 120˚C,

indicated triterpenoid nature of these compounds (Figure 59A). The total extract was

subjected to enzymatic hydrolysis by pectinase and further subjected to TLC analysis

with the same solvent system as above. Except for a spot at Rf 0.88, no spots

corresponding to bands 1, 2 and 3 could be observed. This indicated a possible glycosidic

nature of the compounds present in these bands. Increased activity of hydrolytic enzymes

such as glycosidase and pectinase during storage in fruits and vegetables has been

reported. Hence, it was of interest to determine the stability of the above compounds

during radiation processing and storage. TLC analysis showed a decrease in area of the

major spots (Rf 0.21 and 0.32) with storage in both control and radiation treated samples

(Table 43). The observed decrease could thus be due to the activity of some of the above

enzymes during storage. However, this decrease was significant only beyond 14th day of

storage. No significant radiation specific effect was observed on the above compounds.

250

Figure 59. TLC separation of methanolic extracts obtained from control and irradiated ash

gourd samples stored for various storage periods (A) sprayed with LB spray (B) stained

with iodine vapors

Table 43. Peak area (sq mm) distribution of TLC spots I and 2 as measured by TLC-

densitometry

Different superscripts in a row indicates significant differences between values at p ≤ 0.05

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3.3.2.2. Identification of cucurbitacins

The methanol extract and its hydrolyzate was subjected to TLC analysis using two

different solvent systems (section 2.2.3.5.). (Figure 60)

Figure 60. TLC profiles of unhydrolyzed (spot 1) and hydrolyzed (spot 2) extracts with

different solvent systems (a) Ethyl acetate : benzene (75:25) (b) Chloroform : acetone

(90:10); Cu C – cucurbitacin C; Cu E- cucurbitacin E.

Figure 60 depicts the TLC profile of hydrolyzed and unhydrolyzed methanol extract in

two different solvent systems. The spots corresponding to cucurbitacins (Cu) were

identified by comparing their Rf values with that reported in literature. While the spot at

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Rf value of 0.56 (solvent system 1) and 0.68 (solvent system 2) matched with that of Cu

E, that at Rf 0.37 (solvent system 1) was in agreement with Rf value of standard Cu C

[252, 253]. In order to further confirm their identity, the methanol extract was subjected

to HPLC analysis.

Figure 61 depicts the HPLC separation of the constituents present in the total extract. The

retention time of the two cucurbitacins namely Cu C and Cu E tentatively identified

above was compared with the reported literature value for these compounds. While the

peak at Rt 4.27 min matched with that of Cu E glucoside, no peaks with Rt comparable to

Cu C could be detected in the HPLC profile [254]. In order to further ascertain the

presence of these two compounds, the hydrolyzate of the total extract was subjected to

GC/MS (Figure 62) analysis.

Figure 61. HPLC chromatograms obtained at 254 and 237 nm for identification of

cucurbitacins. (The peak corresponding to Cu E glucoside is marked with arrow).

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Figure 62. GC/MS profile obtained for hydrolyzate of the total extract.

The mass fragmentation (Table 44) of peak 1 and that of peak 2 (Figure 62) was in good

agreement with that of the mass spectra of standard Cu C and CuE respectively , reported

in literature.

The mass spectrum of peak 1 showed a fragment at m/z = 500 (M+

- 60), corresponding to

C30H44O6. Other fragments at m/z 482 (C29H38O6) and 470 (C28H38O6) also matched with

the fragments reported for Cu C. A very intense and characteristic peak, often the base

peak was found at m/z = 43 (CH3CO). The mass fragments at m/z = 113 (C6H8O2) and

m/z = 96 (C6H8O) further indicated the presence of Cu C in the hydrolyzate (Table 44).

On the other hand, mass fragments of peak 2 in Figure 62 matched with that of Cu E (11

AT). The mass spectrum showed a fragment at m/z = 556 (M+) corresponding to

C32H44O8. A very intense and characteristic peak, often the base peak was found at 96

(C6H8O). The mass peak at m/z = 113 might correspond to the formula C6H9O2.

Thus, the aforementioned spectral data and Rf values on TLC (Table 44) indicated the

presence of Cu C and Cu E in the hydrolyzate of total extract. The results obtained are

summarized as below (Table 44)

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Table 44. Rf values, maximum UV absorption and the mass fragments of Cu C and Cu E.

Thus, based on the TLC, HPLC and GC/MS analysis of total and enzymatically

hydrolyzed extract, the major triterpenes were partially identified as Cucurbitacin E

glucoside and Cucurbitacin C (Figure 63). Most of the members of Cucurbitaceae have

been reported to contain less than 0.01 % cucurbitacins [255]. Thus, the structure of the

compounds require further verification, which is in progress.

Cucurbitacin E glucoside Cucurbitacin C

Figure 63. Chemical structure of the compounds identified in this study

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255

3.3.3. Glucosinolates in drumstick

Drumstick (M. oleifera) is an important food commodity which has received enormous

attention as the ‘natural nutrition of the tropics’. Almost all the parts of this plant, viz-

root, bark, gum, leaf, fruit (pods), flowers, seed and seed oil have been used for various

ailments in the indigenous medicine of South Asia, including the treatment of

inflammation and infectious diseases along with cardiovascular, gastro-intestinal,

hematological and hepatorenal disorders [108, 111, 117]. The medicinal properties of the

plant have been attributed to the presence of bioactive phytochemicals including phenolic

compounds, and a unique group of compounds called glucosinolates (GSLs) and

isothiocyanates (ITCs) (58, 113].

Glucosinolates are very stable water-soluble bioactive compounds. Myrosinase (EC

3.2.3.1), a class of enzymes that catalyses the hydrolysis of GSLs, is physically separated

from each other in intact plant cells. Food processing methods such as physical disruption

of plants by chewing, chopping and freezing results in the interaction of GSLs with

myrosinase resulting in the hydrolysis of the latter and liberation of various breakdown

products (isothiocyanates, nitriles, oxazolidinethiones, thiocyanate and epithionitriles)

(Figure 5 in introduction). Under carefully controlled conditions designed to extract

glucosinolates and isothiocyanates completely, while preventing myrosinase activity,

some fresh plants have been shown to contain almost exclusively glucosinolates [60]. In

contrast to intact GSL, hydrolysis products have been of interest for their diverse

biological activities, ranging from antimicrobial, antioxidant and anticancer actions [58].

Among breakdown products, isothiocyanates (ITCs) are reported to have highest

256

biological activity. They have been reported to possess broad-spectrum antimicrobial

activity against bacterial & fungal pathogens, insects and possess potent anticarcinogenic

activity [256, 257]. Bioavailability of these compounds are influenced by storage and

culinary processing [258]. Therefore, the effect of radiation treatment (1 kGy) on

bioactive glucosinolates and isothiocyanates in drumstick was investigated.

3.3.3.1. Isolation and identification of glucosinolates and isothiocyanates

The glucosinolate extract was subjected to HPLC using the method detailed in section

2.2.3.7. The extract was hydrolyzed using the enzymes sulfatase and myrosinase to locate

the peaks corresponding to glucosinolates. Sulfatase removes the sulfo moiety, whereas

myrosinase hydrolyses the sugar moiety and releases free aglycones from the

glucosinolates. The corresponding HPLC profiles are shown in Figure 64.

As evident from Figure 64, the peaks in the initial region (2 – 6 min) of the HPLC

chromatogram of aq extract exhibited quantitative changes when hydrolyzed by

myrosinase and sulfatase. The compounds corresponding to the major peaks in this

region were thus collected for further analysis (Table 45).

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Figure 64. HPLC chromatograms obtained for drumstick aqueous extract and after

hydrolysis with myrosinase and sulfatase enzyme.

Due to unavailability of standard glucosinolates, the total aq extract and the peaks thus

collected were further subjected to enzymatic hydrolysis by myrosinase and the released

aglycones then analyzed by GC/MS in order to identify the corresponding glucosinolates.

Table 46 lists the major compounds detected in the peaks collected from HPLC of the aq

extract. Although, many sulphur containing compounds were detected, no isothiocyanates

could be detected in the collected peaks. The acidic conditions of the eluting solvent

system (presence of TFA in solvent system, section 2.2.3.7.) might have resulted in the

degradation of the isolated peaks resulting in production of sulfones and other acid

258

derivatives instead of isothiocyanates or nitriles. Production of different aglycones via

alternative modes of degradation of glucosinolates under different conditions has been

reported [259]. Therefore, HPLC could not be employed in the present study for

investigating the vaiation in the glucosinolate content as a result of radiation processing

and storage.

Table 45. Major compounds detected in the collected peaks as identified by GC/MS.

Peak No. (Rt in HPLC) Major compounds identified

1 (2.84 min) S-acetyl-2-methyl-3-mercapto propionic acid ester,

carbamidoylsulfanylic acetic acid, dipropyl ester of sulfurous acid

2 (3.14 min) Acetoin, ethyl acetate, dimethyl sulfone, 2,4-Dithiahexan-5-one

3 (3.86 min) Dimethyl sulfone, 2,4-Dithiahexan-5-one, thiolacetic acid, 2,3-

butanediol, cyclic sulfate

4 (4.34 min) 1,3-butanediol, dimethyl sulfone, pyrimidine-2,4(1H, 3H)-dione, 5-

amino-6-nitroso

5 (6.09 min) 2,4-Dithiahexan-5-one, methyl thiomethyl thiol acetate

GC/MS analysis of the hydrolyzate of total aq extract resulted in identification of

isopropyl isothiocyanate, isobutyl isothiocyanate and 2-butyl isothiocyanate as the major

ITCs. GC/MS profile of hydrolyzed aq extract of drumstick is depicted in Figure 65.

Figure 66 represents the chemical structure and mass spectral data of the identified ITCs.

It has been proposed that presence of an ITC or a desulfo-GSL is proof of the parent GSL

[78]. Thus, presence of isopropyl glucosinolate, 2-butyl glucosinolate and isobutyl

glucosinolate in drumstick could be inferred.

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Figure 65: GC/MS profile of hydrolyzed aqueous extract of drumstick

Figure 66. Chemical structure and mass spectra of isopropyl, 2-butyl and isobutyl ITC.

Several researchers have indirectly identified Moringa glucosinolates by analyzing the

free aglycones obtained after myrosinase hydrolysis using GC/MS. These were isopropyl,

isobutyl, 2-methylpropyl, 4-(α-L-rhamnopyranosyloxy)-benzyl and 4’-O-acetyl-4-(α-L-

260

rhamnopyranosyloxy)-benzyl in M. peregrina and isopropyl, 2-methylpropyl, and traces

of isobutyl and 4’-o-acetyl-4-(α-L-rhamnopyranosyloxy)-benzyl in M. pterygosperma,

and 4-(α-L-rhamnopyranosyloxy)-benzyl in M. oleifera and M. stenopetala [113]. Figure

67 shows the structures of glucosinolates previously reported in Moringa species.

Although, isopropyl and isobutyl glucosinolates have been reported earlier in different

species of Moringa, these compounds have not been reported in M. oleifera (drumstick).

Figure 67. Structures of glucosinolates previously reported in Moringa species (adapted

from [113])

In order to study the effect of radiation processing and storage on the glucosinolates in

drumstick, the aq extracts of control and irradiated RTC drumstick at each storage

duration were subjected to hydrolysis by myrosinase. The released isothiocyanates were

then analyzed using GC/MS. Table 46 lists the variation in the contents of major

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isothiocyanates detected in control and radiation treated drumstick at different storage

intervals.

Table 46. Quantitative distribution of major isothiocyanates (µg/kg) in control and

radiation processed (1 kGy) RTC drumstick at different storage periods.

Data are expressed as mean ± standard deviation (n = 6). Mean values in the same column bearing same

superscript shows no significant difference (p ≤ 0.05).

As evident from Table 46, radiation processing resulted in enhanced content of

isothiocyanates. An enhancement of 16, 79 and 64 % in the content of isopropyl, 2-butyl

and isobutyl isothiocyanate respectively was observed in radiation processed (1 kGy)

samples in comparison to non-irradiated controls on day zero. Storage resulted in a

significant decrease in the content of identified ITCs (Table 46). At the end of storage

period (12 d), a decrease of 56, 67 and 65 % was observed in the contents of isopropyl, 2-

butyl and isobutyl isothiocyanate respectively, in control samples. However, this decrease

was only of 19, 44 and 34 % for the respective ITCs in radiation treated samples at the

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end of intended storage period (12 d). Similar reports of decreased ITC content during

storage have been reported in Brassica vegetables [259], where in approximately 30–50%

of the total loss of ITCs in shredded vegetables stored at 4-8 °C was reported. Although,

storage resulted in a significant decrease in the contents of all the three ITCs, this

decrease was appreciably higher in the control than the treated samples at the end of

intended storage period (12 d). Since presence of ITCs has been proposed as a proof of

the existence of the corresponding parent GSLs, the changes in ITCs could be correlated

with the variation in their respective GSLs. The study, thus, demonstrates an increased

content of isopropyl, isobutyl and 2-butyl GSLs, thereby improving the nutraceutical

value of the product, as a result of radiation processing. Radiation induced enhancement

in the glucosinolate content and better retention during storage has also been reported

previously in minimally processed cabbage [145. Upregulation of the genes associated

with GSL biosynthesis was cited as the reason for enhanced contents of GSLs in radiation

processed product. To the best of our knowledge, this is the first report showing effect of

radiation processing on the glucosinolate/ isothiocyanate content of M. oleifera.

3.3.4. Isolation, identification and quantification of phenolic constituents and

carotenoids

Dietary antioxidants have been demonstrated to play a significant health protective role.

Beneficial effects of antioxidants present in fruits and vegetables have been widely

reported [43]. These antioxidants such as polyphenolic compounds including flavonoids

eg. flavonols, flavones have gained considerable interest as they are shown to be very

263

effective in protecting against cardio-vascular and other degenerative diseases [260].

Thus, there is a general tendency to increase the consumption of fresh fruits and

vegetables, as this can help increase the intake of essential bioactive compounds and

antioxidants. Radiation processing has been reported to increase the availability of these

bioactive compounds due to increased extractability and radiolytic breakdown of their

glycosidic precursors. It was therefore of interest to investigate the nature and content of

phenolic compounds in the selected vegetables and determine the effect of radiation

processing and storage on these constituents.

The representative HPLC chromatograms of phenolic compounds in ash gourd, drumstick

and pumpkin are shown in Figure 68.

Figure 68. HPLC chromatograms obtained from ash gourd, drumstick and pumpkin at

280 nm

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3.3.4.1. Ash gourd: The quantitative distribution of phenolic constituents identified in

ash gourd and their changes during radiation processing and storage are represented in

Table 38 (section 3.2.6.1.4.). The representative chromatogram obtained from HPLC is

shown in figure 68. The major phenolic constituents identified in ash gourd included

phenolic acids (α-resorcylic, p-hydroxy benzoic, chlorogenic, syringic, p-coumaric acid)

along with catechin and naringenin (Table 38, section 3.2.6.1.4.). High antioxidant

potential of ash gourd fruit has been reported earlier [261]. Different researchers have

also demonstrated protective effects of ash gourd in the renal failures and injury caused

by mercury chloride [100]. The antioxidant attributes of the fruit were ascribed to the

presence of polyphenolics compounds including iso-vitexin, astilbin, catechin and

naringenin. Besides catechin and naringenein, which were identified previously, the other

phenolic acids identified in the present study have not been reported earlier. Radiation

induced increase in α-resorcylic acid and a decrease in the content of chlorogenic and

caffeic acid was observed. Increased α-resorcylic acid was shown in this study (section

3.2.6.1.4.) to prevent browning of irradiated ash gourd during storage. Radiation induced

decrease in phenolic compounds has been reported in various plant produce [169, 170]. In

general, the decrease in antioxidants and phenolic compounds is attributed to the

formation of radiation-induced degradation products or the formation of free radicals.

However, no significant changes in other phenolic constituents was observed as a result of

radiation processing.

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3.3.4.2. Drumstick

The major phenolic constituents identified in drumstick are represented in Table 47. It

included phenolic acids - gallic, protocatechuic, caffeic and o-coumaric acid and

flavonoids – catechin, epicatechin, kaempferol and rutin. Earlier reports on drumstick

pods demonstrated the presence of rutin, however, phenolic acids were not reported by

these workers [113]. Most of the work published on drumstick till date has been mainly

focused on its leaves, seeds and flowers. Reports in literature on the nature of phenolic

constituents in drumstick pods demonstrate the presence of mainly flavonoid glycosides

such as quercetin-3-O-rutinoside (rutin), quercetin-3-O-glucoside with traces of

kaempferol 3-O-(6”-malonyl glucoside) and isorhamnetin 3-O-(6”-malonyl glucoside).

Caffeoylquinic acids (5- and 3- isomers) were detected in stem, leaves and flower with

exception of roots, pods and seeds [262]. To the best of our knowledge, the presence of

gallic, protocatechuic, caffeic and o-coumaric acids in drumstick pods is reported here for

the first time. However these acids were reported earlier in drumstick seed flour by Singh

et al. [263]. No significant changes in the contents of phenolic constituents identified in

the present study were observed as a result of radiation processing and storage. Thus,

radiation processing was found to be amenable for improving the shelf life of RTC

drumstick without any significant changes in the bioactive phenolic compounds of the

vegetable.

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Table 47. Quantitative distribution of identified phenolic constituents (mg/kg) in

drumstick

Values are means ± SD (n = 3); Different letters indicate significant difference at p ≤ 0.05

3.3.4.3. Pumpkin

3.3.4.3.1. Identification and quantification of phenolic constituents

The nature and content of phenolic compounds were studied in control and radiation-

treated samples. Major phenolic compounds identified in the present study include

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syringic, caffeic, protocatechuic, p-hydroxy benzoic, vanillic and ferulic acid along with a

flavanol, quercetin (Table 48). Similar phenolic composition was earlier reported by

Dragovic-Uzelac et al. [264]. Besides, chlorogenic and p-coumaric acid were also

reported by these workers, which were not detected in the present study. A marginal but

statistically significant (p < 0.05) decrease was noted in the contents of protocatechuic (9

%), caffeic (3 %), ferulic acid (13 %) and quercetin (7 %) due to radiation processing. No

significant (p < 0.05) effect of radiation treatment was, however, observed on the content

of all other phenolic constituents identified (Table 48).

A significant (p < 0.05) reduction in the content of all the phenolic compounds was

observed during storage in control samples. The losses ranged from 6 % for vanillic acid

to a maximum reduction of 31 % in the content of protocatechuic acid on day 21.

Increased physiological and microbial spoilage during storage, resulting in enhanced

degradation of sensitive compounds such as phenolic constituents could possibly explain

the decreased content observed. However, radiation-treated samples had a higher content

of phenolic constituents as compared to non-irradiated controls at the end of storage

period of 21 d (Table 49). Better maintenance of physiological and microbial quality in

the radiation-treated samples could have prevented the leaching out of phenolic

compounds and their exposure to air, resulting in lesser oxidation and thus maintaining

their content during storage.

268

Table 48. Quantitative distribution of identified phenolic constituents and carotenoids

(mg/kg) in pumkin

Values are means ± SD (n = 3); Different letters indicate significant difference at p ≤ 0.05

3.3.4.3.2. Identification and quantification of carotenoids

Carotenoids are the natural plant pigments responsible for the bright yellow-orange

colour of pumpkin. Besides, they are known to possess significant antioxidant activity

and a range of other biological activities [265]. Content of β-carotene and lutein, major

carotenoids identified in the present study are shown in Table 49. The representative

HPLC profile obtained for carotenoids is shown in Figure 69.

269

The content of β-carotene and lutein in fresh control pumpkin samples was 4.75 ± 1.21

mg/kg and 7.76 ± 1.26 mg/kg, respectively. No significant effect of radiation processing

was observed on the content of both the carotenoids immediately after irradiation.

Similar observations have been reported in irradiated carrots and cucumber and sliced

tomatoes [87, 266]. However, a significant (p < 0.05) decrease was observed during

storage in both control and irradiated samples (Table 11). The content of beta carotene

and lutein at the end of storage (21 d) was found to be 3.42 ± 0.65 and 5.81 ± 1.39

respectively in control and 2.40 ± 0.26 and 3.51 ± 0.84 mg kg

-1 respectively in radiation-treated

samples. Similar results have been documented for various radiation-treated Indian potato

cultivars by [267]. wherein they reported 50 % reduction in the total carotenoid content

after 6 months of storage. Thus, from the data obtained in the present study, it could be

concluded that the overall nutritional quality of the radiation-treated (1 kGy) product was

better than the corresponding control at the end of the intended storage (21 d).

270

Figure 69. HPLC chromatograms obtained for control and radiation processed pumpkin

(0 d and 21 d) carotenoids (450 nm).

271

3.4. Conclusions

The experimental design approach was successfully employed for optimization of

radiation dose and storage time for obtaining RTC products of ash gourd, drumstick and

pumpkin with acceptable microbial and sensory quality. Third-order polynomial

equations explained the complex interactions among radiation dose, storage duration and

quality attributes of the RTC products. At an optimum dose of 2 kGy, a shelf life of 12 d

for ash gourd; and at a dose of 1 kGy a shelf life of 12 and 21 d for drumstick and

pumpkin respectively was achieved. Radiation-treated products had better nutritional

attributes as compared to the control at the end of storage period. Analysis of developed

products by QDA demonstrated that the processed products had acceptable aroma, taste

and textural properties. The mathematical approach for obtaining process parameters can

be useful for commercial production as the processing conditions can be suitably

optimized based on quality characteristics required, thus reducing severity of treatments.

The study demonstrated the efficacy of gamma irradiation for extending the shelf-life of

minimally processed vegetables.

Free and bound aroma compounds of the selected vegetables – ash gourd, drumstick and

pumpkin were identified. SPME in case of free aroma and SPE for bound aroma were

found to generate better qualitative and quantitative data in all the three vegetables.

Alcohols, aldehydes, ketones and terpenes were the major classes of compounds

identified. Interference with the solvent in the initial regions of chromatograms and losses

of volatiles during evaporation step in SDE restricted the identification of many lower

boiling aroma compounds in SDE.

272

GC-O analysis of the aroma extracts was successfully employed for the identification of

key odorants in the vegetables studied. Acetoin, octanal and nonanal for ash gourd;

benzothiazole, decanal and 2-dcenal in drumstick and a combination of 6Z-nonenal and

2E, 6Z-nonadienal in case of pumpkin were demonstrated to be the key odorants

responsible for the characteristic aroma of the respective vegetable.

To the best of our knowledge, this is the first report on identification of aroma

constituents in these vegetables. As bound aroma glycosides serve as a reservoir of potent

aroma, the data so generated might be useful in exploiting this pool for

enhancing/modifying the overall aroma quality of the vegetables by using suitable

processing steps. Information about key odorants may be utilized in reconstitution of the

aroma formulations, which can be utilized in the food/ flavor industry.

Radiation processing resulted in significant changes in aroma profile of all the three

vegetables studied. However, the changes varied in each vegetable. Contents of alcohols

increased in response to radiation processing and storage in ash gourd and pumpkin

which could be attributed to the radiolytic breakdown of their corresponding glycosidic

precursors.

The contents of major carbonyl compounds decreased with radiation processing and

storage in ash gourd, while a significant increase in the content of hexanal and trans-2-

hexenal was observed in radiation processed drumstick. The increase in aldehydes content

could be attributed to radiation induced lipid radiolysis resulting in release of linolenic

acid and its subsequent conversion to aldehydes via lipoxygenase (LOX) pathway. On the

other hand, conversion of aldehydes to corresponding branched chain alcohols and

273

subsequently to esters could be the reason for decreased contents of aldehydes in ash

gourd. No significant changes were observed in carbonyl compounds of pumpkin in

response to irradiation.

A considerable difference in the effect of radiation processing on the content of

glycosidic precursors was observed among the three vegetables studied. Two types of

radiation effects were observed. Decrease in glycosidic precursors was seen in ash gourd

and pumpkin, while their contents increased in irradiated drumstick. Both radiation

induced increased extractability and degradation was observed. It can be concluded that

both these changes can occur simultaneously and independent of each other. Despite the

changes in the content of some of the aroma compounds during storage and radiation

processing as noted instrumentally and statistically, they were not sufficient to be

observed by the sensory panel. Hence the radiation processed product developed, had

good sensory acceptability at the end of storage period.

Radiation processing inhibited browning in RTC ash gourd during storage. Enhanced

electrolytic leaching due to softening of tissue structure and enhanced PAL activity were

found to play a major role in increased browning of control samples during extended

storage. Radiation induced increase in the content of α-resorcylic acid resulted in

inhibition of ash gourd PPO that subsequently, prevented browning in radiation processed

samples. The current work demonstrated the feasibility of radiation processing as an

effective post harvest processing method in inhibiting surface browning in RTC ash

gourd. Thus, besides being highly effective method of ensuring food safety, γ-irradiation

provides an improved benefit in terms of maintaining visual quality of the product. To the

274

best of our knowledge, this is the first report on radiation induced browning inhibition in

RTC ash gourd.

At the optimum doses employed for shelf-life extension, (2 kGy for ash gourd; 1 kGy for

drumstick and pumpkin), the bioactive principles were not affected significantly. The

plant growth regulating and ACE inhibition capacity of ash gourd was not altered by

radiation treatment. The contents of major isothiocyanates identified in drumstick

(isopropyl isothiocyanate, isobutyl isothiocyanate and 2-butyl isothiocyanate) was 2-3

times higher in radiation treated drumstick as compared to non-irradiated control at the

end of storage period (12 d), thus resulting in improved nutraceutical value of the

vegetable. An increased content of α-resorcylic acid in irradiated ash gourd resulted in

PPO inhibition and consequently browning development on the cut surfaces of the

vegetable. A marginal decrease in the phenolic contents was observed in radiation-treated

pumpkin, whereas in drumstick, they were unaffected. Thus radiation processing was

found to be amenable for extending the shelf life of RTC products of ash gourd,

drumstick and pumpkin without any adverse effect on the bioactive principles. To the best

of our knowledge, this study demonstrates for the first time the effect of radiation

processing on shelf life improvement and various phyto-chemical aspects of RTC ash

gourd, drumstick and pumpkin.

275

CHAPTER 4

SUMMARY

276

The present study aimed at determining the feasibility of radiation processing for

improving the shelf life of ready-to cook (RTC) vegetables of Indian origin. It was also of

interest to determine the changes, if any, in the quality of the processed products as a

result of radiation processing. The vegetables selected for the study were: ash gourd,

drumstick and pumpkin. The main attributes studied included microbial quality, color,

texture, sensory acceptability and nutritional quality. Changes in bioactive compounds

and their precursors during such a treatment were also investigated.

The highlights of the study are summarized below:

Radiation processing of RTC ash gourd, drumstick and pumpkin, packed in

polystyrene trays and wrapped in cling films was demonstrated to improve their

shelf-life when stored at 10 °C

The full factorial experimental design approach was successfully employed for

optimization of radiation dose and storage time for obtaining RTC products of ash

gourd, drumstick and pumpkin with acceptable microbial and sensory quality.

At an optimum dose of 2 kGy a shelf life of 12 d for ash gourd; and at a dose of 1

kGy a shelf life of 12 and 21 d for drumstick and pumpkin respectively was

achieved.

Application of gamma radiation at optimum doses resulted in development of

RTC products with desired microbial and sensory quality.

Radiation-treated products had better nutritional quality (antioxidant potential,

vitamin C, total phenolic & flavonoid content) as compared to the non-irradiated

controls at the end of intended storage period.

277

The use of radiation processing in combination with cling wrap packaging was

shown to establish aerobic conditions within the package, thus inhibiting the

growth of anaerobic pathogens.

The mathematical approach used for obtaining process parameters was

demonstrated to be useful for commercial production as the processing conditions

can be suitably optimized based on quality characteristics required thereby

reducing severity of treatments.

Gamma irradiation inhibited surface browning in RTC ash gourd during storage.

A decreased quinone formation and enzyme (PAL and PPO) activities and an

increased α-resorcylic acid content, a known PPO inhibitor, as a result of radiation

processing was demonstrated to be responsible for the observed inhibition in

browning.

The nature and contents of free and glycosidically bound aroma compounds was

investigated in all the three vegetables. Acetoin, octanal and nonanal in ash gourd;

benzothiazole, decanal and 2E-decenal in drumstick and a combination of 6Z-

nonenal and 2E, 6Z-nonadienal in case of pumpkin were identified as the key

odorants by employing GC-O technique.

The effect of radiation processing and storage on free and bound aroma

constituents was studied using principal component analysis (PCA).

Radiation induced enhancement in the contents of most of the alcohols was

observed in ash gourd and pumpkin. This could be attributed to the radiolytic

breakdown of their corresponding glycosidic precursors. An increase in the

278

content of hexanal and trans-2-hexenal was also observed in radiation processed

drumstick.

Despite the changes in the content of some of the aroma compounds during

storage and radiation processing as noted instrumentally and statistically, these

changes were not sufficient to bring about alteration in the sensory quality. The

radiation processed products developed, had good sensory acceptability at the end

of storage period.

Acetoin glucoside, a major aroma compound in ash gourd, as a plant growth

regulator was demonstrated. Molecular mechanism of this growth promotion was

shown to be due to the upregulation of RanB1 protein (an important regulatory

protein) in tobacco leaf discs treated with acetoin glucoside.

Angiotensin converting enzyme (ACE) inhibitory activity was demonstrated in

ash gourd juice. The compound responsible for the activity was found to be a

small peptide (323 Da), comprising of alanine and valine. The ACE inhibition

activity of the vegetable juice remained unaffected in response to radiation

treatment.

Radiation processing did not cause any significant changes in the content of

cucurbitacin C & cucurbitacin E glucoside, the major triterpenes of pumpkin.

Presence of isopropyl glucosinolate, 2-butyl glucosinolate and isobutyl

glucosinolate in Moringa oleifera was shown for the first time. Radiation induced

enhancement in the contents of these compounds at the end of storage (12 d) was

demonstrated.

279

No significant effect of radiation treatment was observed on the major phenolic

constituents identified in the selected vegetables.

Radiation processing was found to be amenable for extending the shelf life of

RTC products of ash gourd, drumstick and pumpkin without any adverse effect on

the bioactive principles.

To the best of our knowledge, this study demonstrates for the first time the effect

of radiation processing on shelf life improvement and various phyto-chemical

aspects of RTC ash gourd, drumstick and pumpkin.

280

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List of Publications arising from the thesis

Journal

a. Published:

1. Sharma, J., Chatterjee, S., Kumar, V., Variyar, P. S., & Sharma, A. (2010). Analysis

of free and glycosidically bound compounds of ash gourd (Benincasa hispida):

Identification of key odorants. Food Chemistry, 122(4), 1327-1332.

2. Tripathi, J., Chatterjee, S., Vaishnav, J., Variyar, P. S., & Sharma, A. (2013). Gamma

irradiation increases storability and shelf life of minimally processed ready-to-cook

(RTC) ash gourd (Benincasa hispida) cubes. Postharvest Biology and Technology, 76,

17-25.

3. Tripathi, J., Gupta, S., Mishra, P. K., Variyar, P. S., & Sharma, A. (2014).

Optimization of radiation dose and quality parameters for development of ready-to-cook

(RTC) pumpkin cubes using a statistical approach. Innovative Food Science & Emerging

Technologies, 26, 248-256.

b. Manuscript under preparation

1. Tripathi, J., Adiani, V., Ghosh, S. B., Ganapathi, T. R., Bauri, A.K., Chatterjee, S.,

Kulkarni, V.M., Variyar, P. S., & Sharma, A. Identification of GTP binding nuclear

protein Ran as an upregulation target in acetoin glucoside mediated plant growth

enhancement

2. Tripathi, J., & Variyar, P. S. Optimization of radiation dose and quality parameters for

development of ready-to-cook (RTC) drumstick using a statistical approach.

317

3. Tripathi, J. & Variyar, P. S. Mechanism of browning inhibition in radiation processed

ready-to-cook ash gourd.

4. Tripathi, J., & Variyar, P. S. Analysis of free and glycosidically bound compounds of

drumstick (Moringa oleifera) and pumpkin (Cucurbita pepo): Identification of key

odorants.

Conferences

1. Sharma, J., Vivekanand Kumar, Chatterjee,S., Variyar,P. S. and Sharma, A.

Identification of aroma impact compounds of ash gourd: Proceedings of the 13th

ISMAS-WS on Mass Spectrometry,(2008),184-190,ISBN:978-81-904442-1-7.

2. Sharma, J., Chatterjee, S., Gupta, S., Kumar, V., Variyar, P. S. and Sharm, A.

Development of shelf stable radiation processed ready-To-Cook (RTC) Indian

vegetables. Peaceful Uses of Atomic Energy, 29th Sep-1st Oct. 2009 held at Vigyan

bhawan, New Delhi.

3. Tripathi, J., Variyar, P. S. and Sharma, A. Mechanism of browning inhibition in

radiation processed ready-to-cook ash gourd. Poster presented at DAE-BRNS Life

Science Symposium (LSS) 2012, "Trends in Plant, Agriculture and Food Science"

(TIPAFS) at multipurpose hall, Training School Hostel, Anushakti Nagar, Mumbai, Dec,

17-19 2012.

4. Tripathi, J., Gupta, S., Variyar, P. S. and Sharma, A. Improving shelf life of gamma

radiation processed Ready-to-cook (RTC) pumpkin cubes: A multivariate statistical

318

approach. Poster presented at IFCON-2013, 18-21 December, 2013 held at CSIR-

CFTRI, Mysore.

5. Tripathi, J., Gupta, S., Variyar, P. S. and Sharma, A. Improving shelf life of gamma

radiation processed Ready-to-cook (RTC) drumstick: A multivariate statistical approach.

Poster presented at LSS-2015, 3-5 Feb, 2015 held at NUB, Anushakti Nagar, Mumbai.

Others

1. Sharma, J., Chatterjee, S., Gupta, S., Kumar, V., Variyar, P. S. and Sharma, A.

Development of shelf-stable radiation processed Ready-to-cook (RTC) Indian

vegetables. Thematic volume evolved from the proceedings of International Conference

on Peaceful uses of atomic energy-2009, Sep 29- Oct 1, 2009, Vigyan Bhavan, New

Delhi, (2011), 154-158.

2. Tripathi, J., Gupta, S., Kumar, V., Chatterjee, S., Variyar, P. S and Sharma, A.

Processing food for Convenience: Challenges and Potentials. BARC Newsletter, (2011),

322, 55-60.

Jyoti Tripathi

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